BackgroundTranscriptomics analyses of bacteria (and other organisms) provide global as well as detailed information on gene expression levels and, consequently, on other processes in the cell. RNA sequencing (RNA-seq) has over the past few years become the most accurate method for global transcriptome measurements and for the identification of novel RNAs. This development has been accompanied by advances in the bioinformatics methods, tools and software packages that deal with the analysis of the large data sets resulting from RNA-seq efforts.ResultsBased on years of experience in analyzing transcriptome data, we developed a user-friendly webserver that performs the statistical analysis on the gene expression values generated by RNA-seq. It also provides the user with a whole range of data plots. We benchmarked our RNA-seq pipeline, T-REx, using a case study of CodY mutants of Bacillus subtilis and show that it could easily and automatically reproduce the statistical analysis of the cognate publication. Furthermore, by mining the correlation matrices, k-means clusters and heatmaps generated by T-REx we observed interesting gene-behavior and identified sub-groups in the CodY regulon.ConclusionT-REx is a parameter-free statistical analysis pipeline for RNA-seq gene expression data that is dedicated for use by biologists and bioinformaticians alike. The tables and figures produced by T-REx are in most cases sufficient to accurately mine the statistical results. In addition to the stand-alone version, we offer a user-friendly webserver that only needs basic input (http://genome2d.molgenrug.nl).Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1834-4) contains supplementary material, which is available to authorized users.
Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.
Here, we developed a cell-based biosensor that can assess meat freshness using the Gram-positive model bacterium Bacillus subtilis as a chassis. Using transcriptome analysis, we identified promoters that are specifically activated by volatiles released from spoiled meat. The most strongly activated promoter was PsboA, which drives expression of the genes required for the bacteriocin subtilosin. Next, we created a novel BioBrick compatible integration plasmid for B. subtilis and cloned PsboA as a BioBrick in front of the gene encoding the chromoprotein amilGFP inside this vector. We show that the newly identified promoter could efficiently drive fluorescent protein production in B. subtilis in response to spoiled meat and thus can be used as a biosensor to detect meat spoilage.
Previous RNA sequencing has allowed identifying 129 long 5'-UTRs in the L. lactis MG1363 transcriptome. These sequences potentially harbor cis -acting riboswitches. One of the identified extended 5'-UTRs is a putative thiamine pyrophosphate (TPP) riboswitch. It is located immediately upstream of the thiamine transporter gene thiT ( llmg_0334 ). To confirm this assumption, the 5'-UTR sequence was placed upstream of the gene encoding the super folder green fluorescent protein, sfgfp , allowing examining the expression of sfGFP in the presence or absence of thiamine in the medium. The results show that this sequence indeed represents a thiamine-responsive TPP riboswitch. This RNA-based genetic control device was used to successfully restore the mutant phenotype of an L. lactis strain lacking the major autolysin gene acmA . The L. lactis thiT TPP riboswitch (RS thiT ) is a useful molecular genetic tool enabling to gradually downregulate the expression of genes under its control by adjusting thiamine concentration. Importance The capacity of microbes with biotechnological importance to adapt and survive under quickly changing industrial conditions depends on their ability to adequately control gene expression. Riboswitches are important RNA-based elements involved in rapid and precise gene regulation. Here we present the identification of a natural thiamine-responsive riboswitch of Lactococcus lactis , a bacterium used worldwide in the production of dairy products. We used it to restore a genetic defect in an L. lactis mutant and show that it is a valuable addition to the ever-expanding L. lactis genetic toolbox.
The Eocene Monllobat Formation of the southern Pyrenees accumulated under continental (non-marine) conditions. The total thickness of 180 m consists of seven cycles, defined by coarse member concentrations (often sheets) at the bases. The lateral extent of the sheets defines the fixed positions of eight parallel-trending alluvial systems, separated by narrow zones of fine sediment.Sheetflooding dominated the sedimentation of these eight contemporaneous alluvial systems. The sediment was supplied from the rising axial zone of the Pyrenees. The cycles were caused by variations in tectonic activity. The positions and forms of coarse members were locally determined by contemporaneous faulting and folding of the alluvium.
The Eocene Monllobat and Castigaleu Formations in the Southern Pyrenees consist of continental and shallow marine deposits respectively. The Monllobat Formation contains conglomerate sheets with sandstone tails, symmetrical channel fills with wings and various types of lobes, accreted laterally in channels as well as by progradation. These deposits are intercalated in large amounts of flat-bedded silt and mudstone with mottling and caliche.The high energy alluvial flow, which deposited these sediments, was initially at right angles to the axis of the elongated basin, becoming near parallel to the axis distally. The deposits are less gravelly distally. Upstream wide, gravelly braidplains were developed. Gravel lobes and muddy sheetfloods prograded over the distal floodplain. Meandering rivers were restricted to a marginal position in the sheetflood environment.The Castigaleu Formation consists of mudstone with intercalations of sandstone. Lobes of conglomerate and sandstone dominate the contact zone with the Monllobat Formation. Flat sandstone layers with tabular cross beds and parallel lamination and massive sandstone beds are distributed throughout the other parts.The lobes were formed at the coastline during high energy inflow in the marine basin. Flow deceleration caused the deposition of the coarsest load at this site, building a wedge with an inclined front during major aggradation and progradation. The other deposition types originated during hyperpycnal inflow in a marine environment. Massive sandstones originated partly because of bioturbation. Reworking by physical marine processes did not occur. Synsedimentary tectonics determined the type of sedimentation at times.
Van der Meulen, S., 1983. Internal structure and environmental reconstruction of Eocene transitional fan-delta deposits, Monllobat-Castigaleu formations, Southern Pyrenees, Spain. Sediment. Geol., 37: 85-112.A detailed study has been made of a part (500 × 200 × 15 m) of the Eocene Monllobat Formation. The conglomeratic bottomset-foreset-topset build up of a small fan-delta passes into sand-and mudstone layers. Ultimately the sandstone layers wedge into the mudstone due to concave-upwards basal surfaces and convex top surfaces or due to thickening of intercalated mud layers. Sequences vary accordingly from coarsening upwards in the proximal parts to fining upwards distally. Both sequences are topped by a thin layer fining upwards.The proximal, fan-deltaic part consists of interconnected lobes. Crescentic, transverse bars deposited gravel on a slightly inclined top. Conglomerates of the moderately inclined foreset show some imbrication. The bottomset consists of tabular cross-bedded and cross-laminated sandstone. Parallel-laminated and tabular cross-bedded sandstone layers drape rises of fine sediment in front of the fan-delta face. Continued progradation brought about truncation of these backset sediments.Sandstone lobes of the distal part are not connected. Tabular cross-bedding and parallel lamination are the main sedimentary structures. Climbing, large-scale bedforms are prominent on top surfaces. Most of the lobes are enveloped by blue-grey mud. Layers covered by mottled mud terminate in foreset faces.Highly energetic inflow took place from a shallow, braided stream with longitudinal bars. The low sedimentary refief caused jetflow. The high flow energy, the large grain-size range and the large quantities of sediment caused great differences from inflow models for the deltaic environment. Sedimentation was effected by flow deceleration. Expansion, inertial forces, friction or a combination of these phenomena were responsible for the deceleration. Expansion and inertial forces caused development of moderately inclined foreset faces. Due to expansion, steeply inclined foresets were formed. Sand was deposited in shallow marine water and in scour pits in continental deposits during flow deceleration caused by frictional and inertial forces. Parallel-laminated backsets connected with flat, tabular sets developed due to flow deceleration by expansion and frictional forces.The deposits were formed in the transition zone of a fan-delta under a tropical chmate with marked dry and wet periods. Wide, braided streams entered a restricted, shallow marine basin at right angles to the basin axis. Aggradation exceeded water depth. Progradation processes were influenced by large-and small-scale tectonic activity.
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