RNA-seq of wild type Mycobacterium smegmatis mc2 155 (exponential and stationary phase) and Ms1 deletion strain (exponential and stationary phase)
ABSTRACT: Ms1 RNA is ~300 nt sRNA that is highly expressed in stationary phase of growth and binds to the RNA polymerase (RNAP) core. We assume that by binding to RNAP, Ms1 could regulate transcription. Our aim was to reveal the most prominent changes in the transcriptome upon entry into stationary phase that might be dependent on Ms1. We performed RNA-seq data to characterize exponential (Ms_WT_exp) and stationary phase (Ms_WT_stat) transcriptome in M. smegmatis in wild type cells. In addition, we compared the transcriptome of the Ms1 knockout cells (Ms_KO_stat) with wild type cells in stationary phase and in exponential phase (Ms_KO_exp).
Project description:Ms1 RNA is ~300 nt sRNA that is highly expressed in stationary phase of growth and binds to the RNA polymerase (RNAP) core. We assume that by binding to RNAP, Ms1 could regulate transcription. Our aim was to reveal the most prominent changes in the transcriptome upon entry into stationary phase that might be dependent on Ms1. We performed RNA-seq data to characterize exponential (Ms_WT_exp) and stationary phase (Ms_WT_stat) transcriptome in M. smegmatis in wild type cells. In addition, we compared the transcriptome of the Ms1 knockout cells (Ms_KO_stat) with wild type cells in stationary phase and in exponential phase (Ms_KO_exp).
Project description:Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (?A) or any other ? factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of ?A. The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with ?A. Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.
Project description:The transition between exponential and stationary phase is a natural phenomenon for all bacteria and requires a massive readjustment of the bacterial transcriptome. Exoribonucleases are key enzymes in the transition between the two growth phases. PNPase, RNase R and RNase II are the major degradative exoribonucleases in Escherichia coli. We analysed the whole transcriptome of exponential and stationary phases from the WT and mutants lacking these exoribonucleases (?pnp, ?rnr, ?rnb, and ?rnb?rnr). When comparing the cells from exponential phase with the cells from stationary phase more than 1000 transcripts were differentially expressed, but only 491 core transcripts were common to all strains. There were some differences in the number and transcripts affected depending on the strain, suggesting that exoribonucleases influence the transition between these two growth phases differently. Interestingly, we found that the double mutant RNase II/RNase R is similar to the RNase R single mutant in exponential phase while in stationary phase it seems to be closer to the RNase II single mutant. This is the first global transcriptomic work comparing the roles of exoribonucleases in the transition between exponential and stationary phase.
Project description:Growth phase dependent transcriptional and translational changes of Hb. salinarum were examined. In the genome-wide transcriptome analysis, gene expression in exponential versus stationary growth phase was monitored on DNA microarrays. In the global study of translational regulation, the relative amounts of free versus polysome-bound mRNA (separated by gradient centrifugation) were quantified in exponential as well as stationary growth phase using DNA microarrays.
Project description:Comparison of Bacillus subtilis wild type and cshA mutant at exponential versus stationary phase. Detailed description (other than provided below) of growth conditions, RNA preparation, cDNA synthesis and hybridization conditions can be found in the submitted paper. Bacillus subtilis 168 wild type and its cshA derivative strain were grown in CSE-Glu medium and cells were harvested at mid exponential (OD600 ~0.8) and early stationary phase (OD600 ~2.2-2.4). For both time points, 4 biological replicates were used and 2 were mixed after cDNA labeling, resulting 2 slides for both exponential and stationary phase. Dye swaps are included in both experiments.
Project description:In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is "compacted" or "supercompacted," and there are suggestions that the cytoplasm is "glass-like." Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery.IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.
Project description:Transcriptional profiling of Salmonella Typhimurium strains SL1344 in exponential and stationary phase cultures Overall design: The transcriptome of wild-type Salmonella Typhimurium SL1344 was determined in cultures grown in LB (Lennox broth) to exponential phase (OD 0.2) and stationary phase (OD 2.0)
Project description:Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation.