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.