Whole genome mapping of 5' RNA ends in bacteria by tagged sequencing : A comprehensive view in Enterococcus faecalis

Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first comprehensive view of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5'RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites and 352 processing sites in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding TSSs mapped. We discovered 95 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organisations. Presented data constitute a significant insight into bacterial RNA landscapes and a step towards the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner

A simple and efficient method to search for selected primary transcripts: non-coding and antisense RNAs in the human pathogen Enterococcus faecalis

Enterococcus faecalis is a commensal bacterium and a major opportunistic human pathogen. In this study, we combined in silico predictions with a novel 5′RACE-derivative method coined ‘5′tagRACE’, to perform the first search for non-coding RNAs (ncRNAs) encoded on the E. faecalis chromosome. We used the 5′tagRACE to simultaneously probe and characterize primary transcripts, and demonstrate here the simplicity, the reliability and the sensitivity of the method. The 5′tagRACE is complementary to tiling arrays or RNA-sequencing methods, and is also directly applicable to deep RNA sequencing and should significantly improve functional studies of bacterial RNA landscapes. From 45 selected loci of the E. faecalis chromosome, we discovered and mapped 29 novel ncRNAs, 10 putative novel mRNAs and 16 antisense transcriptional organizations. We describe in more detail the oxygen-dependent expression of one ncRNA located in an E. faecalis pathogenicity island, the existence of an ncRNA that is antisense to the ncRNA modulator of the RNA polymerase, SsrS and provide evidences for the functional interplay between two distinct toxin–antitoxin modules

Analysis of the Peptidoglycan Hydrolase Complement of Lactococcus lactis: Identification of a Third N-Acetylglucosaminidase, AcmC

The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in L. lactis subsp. cremoris MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity

Peptidoglycan Structure Analysis of Lactococcus lactis Reveals the Presence of an l,d-Carboxypeptidase Involved in Peptidoglycan Maturation

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation

Dynamic insights on transcription initiation and RNA processing during bacterial adaptation

International audienceTranscription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously-described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity; i) constant and independent of RNA levels, ii) increased once RNA has accumulated, and iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes, and can serve as blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen <i>Enterococcus faecalis</i>

<p>We discovered a chromosomal locus containing 2 toxin-antitoxin modules (TAs) with an antisense transcriptional organization in the E. faecalis clinical isolate V583. These TAs are homologous to the type I txpA-ratA system and the type II mazEF, respectively. We have shown that the putative MazF is toxic for E. coli and triggers RNA degradation, and its cognate antitoxin MazE counteracts toxicity. The second module, adjacent to mazEF, expresses a toxin predicted to belong to the TxpA type I family found in Firmicutes, and the antisense RNA antidote, RatA. Genomic analysis indicates that the cis-association of mazEF and txpA-ratA modules has been favored during evolution, suggesting a selective advantage for this TA organization in the E. faecalis species. We showed regulatory interplays between the 2 modules, involving transcription control and RNA stability. Remarkably, our data reveal that MazE and MazEF have a dual transcriptional activity: they act as autorepressors and activate ratA transcription, most likely in a direct manner. RatA controls txpA RNA levels through stability. Our data suggest a pivotal role of MazEF in the coordinated expression of mazEF and txpA-ratA modules in V583. To our knowledge, this is the first report describing a crosstalk between type I and II TAs.</p