GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

RNase J depletion leads to massive changes in mRNA abundance in Helicobacter pylori.

Degradation of RNA as an intermediate message between genes and corresponding proteins is important for rapid attenuation of gene expression and maintenance of cellular homeostasis. This process is controlled by ribonucleases that have different target specificities. In the bacterial pathogen Helicobacter pylori, an exo- and endoribonuclease RNase J is essential for growth. To explore the role of RNase J in H. pylori, we identified its putative targets at a global scale using next generation RNA sequencing. We found that strong depletion for RNase J led to a massive increase in the steady-state levels of non-rRNAs. mRNAs and RNAs antisense to open reading frames were most affected with over 80% increased more than 2-fold. Non-coding RNAs expressed in the intergenic regions were much less affected by RNase J depletion. Northern blotting of selected messenger and non-coding RNAs validated these results. Globally, our data suggest that RNase J of H. pylori is a major RNase involved in degradation of most cellular RNAs.

MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data.

MicroScope is an integrated platform dedicated to both the methodical updating of microbial genome annotation and to comparative analysis. The resource provides data from completed and ongoing genome projects (automatic and expert annotations), together with data sources from post-genomic experiments (i.e. transcriptomics, mutant collections) allowing users to perfect and improve the understanding of gene functions. MicroScope (http://www.genoscope.cns.fr/agc/microscope) combines tools and graphical interfaces to analyse genomes and to perform the manual curation of gene annotations in a comparative context. Since its first publication in January 2006, the system (previously named MaGe for Magnifying Genomes) has been continuously extended both in terms of data content and analysis tools. The last update of MicroScope was published in 2009 in the Database journal. Today, the resource contains data for >1600 microbial genomes, of which ∼300 are manually curated and maintained by biologists (1200 personal accounts today). Expert annotations are continuously gathered in the MicroScope database (∼50 000 a year), contributing to the improvement of the quality of microbial genomes annotations. Improved data browsing and searching tools have been added, original tools useful in the context of expert annotation have been developed and integrated and the website has been significantly redesigned to be more user-friendly. Furthermore, in the context of the European project Microme (Framework Program 7 Collaborative Project), MicroScope is becoming a resource providing for the curation and analysis of both genomic and metabolic data. An increasing number of projects are related to the study of environmental bacterial (meta)genomes that are able to metabolize a large variety of chemical compounds that may be of high industrial interest.

The genomic and transcriptomic basis of the potential of Lactobacillus plantarum A6 to improve the nutritional quality of a cereal based fermented food.

The objective of this work was to investigate the nutritional potential of Lactobacillus plantarum A6 in a food matrix using next generation sequencing. To this end, we characterized the genome of the A6 strain for a complete overview of its potential. We then compared its transcriptome when grown in a food matrix made from pearl millet to and its transcriptome when cultivated in a laboratory medium. Genomic comparison of the strain L. plantarum A6 with the strains WCFS1, ST-III, JDM1 and ATCC14917 led to the identification of five regions of genomic plasticity. More specifically, 362 coding sequences, mostly annotated as coding for proteins of unknown functions, were specific to L. plantarum A6. A total of 1201 genes were significantly differentially expressed in laboratory medium and food matrix. Among them, 821 genes were up-regulated in the food matrix compared to the laboratory medium, representing 23% of whole genomic objects. In the laboratory medium, the expression of 380 genes, representing 11% of the all genomic objects was at least double than in the food matrix. Genes encoding important functions for the nutritional quality of the food were identified. Considering its efficiency as an amylolytic strain, we investigated all genes involved in carbohydrate metabolism, paying particular attention to starch metabolism. An extracellular alpha amylase, a neopullulanase and maltodextrin transporters were identified, all of which were highly expressed in the food matrix. In addition, genes involved in alpha-galactoside metabolism were identified but only two of them were induced in food matrix than in laboratory medium. This may be because alpha galactosides were already eliminated during soaking. Different biosynthetic pathways involved in the synthesis of vitamin B (folate, riboflavin, and cobalamin) were identified. They allowed the identification of a potential of vitamin synthesis, which should be confirmed through biochemical analysis in further work. Surprisingly, some genes involved in metabolism and bioaccessibility of iron were identified. They were related directly to the use of transport of iron, or indirectly to metabolism of polyphenols, responsible of iron chelation in the food. The combination of genomics and transcriptomics not only revealed previously undocumented nutritional properties of L. plantarum A6, but also documented the behaviour of this bacterium in food.

Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium.

Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens, by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens. Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in either strain CM4 or strain DM4 total 13 and 58 CDS, respectively. Taken together, the obtained results suggest different transcriptional responses of chloromethane- and dichloromethane-degrading M. extorquens strains to dehalogenative metabolism, and substrate- and pathway-specific modes of growth optimization with chlorinated methanes.