Analyses of competent and non‐competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence
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ABSTRACT:
PROVIDER: PRJNA726040 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14355156.fastq.gz | Fastqsanger.gz | |||
| SRR14355157.fastq.gz | Fastqsanger.gz | |||
| SRR14355158.fastq.gz | Fastqsanger.gz | |||
| SRR14355159.fastq.gz | Fastqsanger.gz | |||
| SRR14355160.fastq.gz | Fastqsanger.gz |
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Project description:Upon competence‐inducing nutrient‐limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence‐assisted cell sorting (FACS). Using RNA‐seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post‐transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK‐binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA‐seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.
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Project description:Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.
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Project description:Cumulus cells surrounding the oocyte were sampled at the following stages: developmentally incompetent or poorly competent prophase I oocytes (NC1 oocytes), developmentally competent prophase I oocytes (C1 oocytes), and developmentally competent metaphase II oocytes (C2 oocytes). NC1 samples were collected from immature, unexpanded cumulus-oocytes complexes (COC) from prepubertal (3-week-old) mice, C1 samples from immature, unexpanded cumulus-oocytes complexes (COC) from adult (8-week-old) and C2 samples from mature, expanded COCs obtained from the oviduct from 8-week-old mice after standard superovulation protocol. Global transcriptional profiling was performed using cumulus cells collected from murine ovarian follicles during in vivo oocyte developmental competence acquisition. Cumulus cells were collected at 3 stages: early stage follicles (prophase I arrested oocytes, meiotically competent but developmentally incompetent, n=5), late stage follicles (prophase I arrested oocytes, meiotically competent and developmentally competent, n=5) and ovulatory follicles collected in vivo (metaphase II arrested oocytes, developmentally fully competent, n=5).
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Project description:Somatic cells surrounding the oocyte were sampled at the following stages: developmentally incompetent or poorly competent prophase I oocytes (NC1 oocytes), developmentally competent prophase I oocytes (C1 oocytes), and developmentally competent metaphase II oocytes (C2 oocytes). NC1 samples were collected from late vitellogenic females (LV), C1 samples from post-vitellogenic females (PV), and C2 samples from females undergoing meiotic maturation (Germinal Vesicle Breakdown) Global transcriptional profiling was performed using somatic cells collected from rainbow trout ovarian follicles during in vivo oocyte developmental competence acquisition. Somatic cells were collected at 3 stages of oogenesis: NC1 stage follicles (LV, late vitellogenic, prophase I arrested oocytes, meiotically incompetent and developmentally incompetent, n=6), C1 stage follicles (PV, post-vitellogenic, prophase I arrested oocytes, meiotically competent and developmentally competent, n=16). Ovulatory follicles were also collected during oocyte maturation after in vivo induction (metaphase II arrested oocytes, developmentally fully competent, n=6).
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Project description:Transcriptional profiling of the competence-stimulating peptide (CSP) response in Streptococcus mutans of FACS-separated subpopulations and mixed cells of a clonal culture. CSP-mediated competence development in S. mutans is a transient and biphasic process since only a subpopulation induces expression of ComX in the presence of CSP and activation of the DNA uptake machinery in this fraction shuts down ~3-4 hours post induction. Here we combine, for the first time in bacteria to our knowledge, flow cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and non-competent fractions of CSP-treated S. mutans cells. Sorting was guided by a ComX-GFP reporter and the transcriptome analysis demonstrated the successful combination of both methods because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, among them ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. By contrast, expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and mutacin V confirmed this expression pattern on the single cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, uptake of DNA could be shown on the single cell level. This study demonstrates that all cells in the population respond to CSP through activation of bacteriocin-related genes but that two subpopulations segregate, one becoming competent and another one that lyses, resulting in intrapopulation diversity of the clonal culture. Two conditions: induced and not induced with CSP. Three induced cell populations: competent subpopulation, incompetent subpopulation, all cells. Two biological replicates each population.
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