RESUMEN
Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot 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 (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.
Asunto(s)
Regiones no Traducidas 5'/genética , Mapeo Cromosómico/métodos , Enterococcus faecalis/genética , ARN Bacteriano/genética , Análisis de Secuencia de ARN/métodos , Lugares Marcados de Secuencia , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Desnaturalización de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN , Sitio de Iniciación de la Transcripción , TranscriptomaRESUMEN
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.
Asunto(s)
Antitoxinas/genética , Toxinas Bacterianas/genética , Enterococcus faecalis/genética , ARN sin Sentido/genética , Secuencia de Aminoácidos , Proteínas de Unión al ADN/genética , Endorribonucleasas/genética , Enterococcus faecalis/patogenicidad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Estabilidad del ARN/genéticaRESUMEN
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.
Asunto(s)
Enterococcus faecalis/genética , ARN sin Sentido/genética , ARN no Traducido/genética , Análisis de Secuencia de ARN , Toxinas Bacterianas/genética , Secuencia de Bases , Secuencia Conservada , Enterococcus faecalis/metabolismo , Sitios Genéticos , Estrés Oxidativo , Péptidos/genética , ARN sin Sentido/análisis , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN no Traducido/análisis , ARN no Traducido/metabolismo , Lugares Marcados de SecuenciaRESUMEN
Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Péptidos Cíclicos/biosíntesis , Percepción de Quorum , Streptococcus thermophilus/genética , Streptococcus thermophilus/fisiología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida , Genes Bacterianos , Espectrometría de Masas , Mutación , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Péptidos Cíclicos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Análisis de Secuencia de Proteína , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Carboxipeptidasas/metabolismo , Lactococcus lactis/enzimología , Peptidoglicano/química , Peptidoglicano/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Amplificación de Genes , Péptidos/química , Péptidos/aislamiento & purificación , Peptidoglicano/genéticaRESUMEN
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.
Asunto(s)
Acetilglucosaminidasa/metabolismo , Prueba de Complementación Genética , Lactococcus lactis/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/genética , Acetilglucosaminidasa/química , Acetilglucosaminidasa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/genética , Lactococcus lactis/crecimiento & desarrollo , Datos de Secuencia Molecular , Peptidoglicano/metabolismo , Análisis de Secuencia de ADNRESUMEN
A gene encoding a putative peptidoglycan hydrolase, named acmB, which is a paralogue of the major autolysin acmA gene, was identified in the Lactococcus lactis genome sequence. The acmB gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth. The encoded AcmB protein has a modular structure with three domains: an N-terminal domain, especially rich in Ser, Thr, Pro and Asn residues, resembling a cell-wall-associated domain; a central domain homologous to the Enterococcus hirae muramidase catalytic domain; and a C-terminal domain of unknown function. A recombinant AcmB derivative, devoid of its N-terminal domain, was expressed in Escherichia coli. It exhibited hydrolysing activity on the peptidoglycan of several Gram-positive bacteria, including L. lactis. Though showing sequence similarity with enterococcal muramidase, AcmB has N-acetylglucosaminidase specificity. The acmB gene was inactivated in order to evaluate the role of the enzyme. AcmB does not appear to be involved in cell separation but contributes to cellular autolysis.