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ABSTRACT: NusG-dependent transcription pausing in Bacillus subtilis

PROVIDER: PRJNA603835 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10987365.fastq.gz	Fastqsanger.gz
	SRR10987366.fastq.gz	Fastqsanger.gz
	SRR10987367.fastq.gz	Fastqsanger.gz
	SRR10987368.fastq.gz	Fastqsanger.gz
	SRR10987369.fastq.gz	Fastqsanger.gz

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Transcriptome-wide effects of NusA on RNA polymerase pausing in Bacillus subtilis

Project description:In this work we conducted RNET-seq on Wild Type, NusA depletion, nusG deletion, and NusA depletion nusG deletion strains of B. subtilis. Using this data we analysed the transcriptome-wide effects of NusA on RNA polymerase pausing and found that NusA has modest pause stimulating as well as suppressing effects throughout the genome.

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NusG is an intrinsic transcription termination factor that stimulates motility and cooperatively coordinates global gene expression with NusA

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2021-04-19 | GSE154522 | GEO

Bacillus velezensis LoaP Promotes Antitermination of Antibiotic Synthesis Operons by Antagonizing NusA

Project description:The transcription factor NusG is essential for the bacterial transcription elongation complex (TEC). During elongation, NusG dynamically interacts with other proteins, affecting transcription-translation coupling, processivity, and termination. NusG is integral to antitermination complexes, where it associates with other factors to modify the TEC such that it bypasses termination sites. In Gammaproteobacteria, these complexes enhance the expression of ribosomal RNA operons, bacteriophage operons, and CRISPR arrays. Specialized NusG paralogs, such as LoaP, RfaH, and UpxY, are believed to form their own unique antitermination complexes. RfaH, the only well-studied paralog, binds to a paused TEC via a specific nontemplate DNA sequence. The RfaH-bound TEC then enhances transcription-translation coupling while also preventing Rho termination. However, it is unclear whether the other NusG paralogs use similar mechanisms, nor have antitermination complexes been investigated for Gram-positive bacteria. Previously, Bacillus velezensis LoaP was shown to enhance transcription of the difficidin antibiotic synthesis operon, but its antitermination determinants were unknown. In this study, we purified RNA polymerase and transcription factors, and reconstituted difficidin operon (dfn) antitermination activity in vitro. The dfn 5’ leader region contains a small RNA hairpin essential for antitermination in vivo and in vitro, and an intrinsic terminator that is highly dependent on NusA. The antitermination activity of LoaP emanates from its ability to specifically antagonize NusA; no other factors are required. These findings demonstrate the basic requirements of the LoaP class of NusG paralogs and indicate that the LoaP antitermination mechanism differs significantly from that of the RfaH paradigm.

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Project description:In this work we conducted Term-seq on Wild Type B. subtilis, and strains compromosing all logical mutations in all combinations of the genes that encode for NusA, NusG, and Rho, where we developed the first transcription termination atlas. We used this atlas to find that Rho can stimulate intrinsic termination in B. subtilis and we recapitulated this finding in vitro to dissect the mechanism.

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Project description:To obtain an insight into the in vivo dynamics of RNA polymerase (RNAP) on the B. subtilis genome, we analyzed the distribution of ?A and ? subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells, using the ChAP (Chromatin Affinity Precipitation)-chip method. In contrast to E. coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences in the majority of genes. This finding suggests that B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which are attributed to transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior during initiation and early elongation steps between E. coli and B. subtilis result in distinct strategies for post-initiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis. Wild-type strain, Bacillus subtilis 168, was also used for RNA and genomic DNA extraction and analysis - RNA data was divided by genome DNA data to normalize (1) PCR bias and (2) copy number of RNA molecule per genome for multi-copy genome of exponentially growing bacteria.

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Project description:The aim of this study was to explore whether, and if so, how Bacillus subtilis KC1 can enhance the growth performance of broilers that have been adversely affected by Mycoplasma gallisepticum (MG) infection. A total of 96 1-day-old male broilers were randomly divided into 4 groups: the control group (basal diet), the MG group (basal diet + MG challenge), the Bacillus subtilis KC1 group (basal diet + Bacillus subtilis KC1 supplementation), the Bacillus subtilis KC1 + MG group (basal diet + Bacillus subtilis KC1 supplementation + MG challenge). The trial lasted 42 days, and the results showed that the MG group had significantly reduced body weight and average daily gain, as well as increased feed conversion ratio of broilers, compared to the control group. Dietary supplementation with Bacillus subtilis KC1 significantly improved the growth performance of MG-infected broilers. In addition, dietary supplementation with Bacillus subtilis KC1 significantly improved oxidative stress and inflammatory response markers, characterized by increased superoxide dismutase levels and reduced levels of malondialdehyde, interleukin-1β, and tumor necrosis factor-α. Furthermore, both metabolomics and transcriptomics analyses indicated that MG infection markedly disrupted amino acid metabolism in broilers, whereas Bacillus subtilis KC1 supplementation alleviated the abnormal amino acid metabolism caused by MG infection. These results suggested that Bacillus subtilis KC1 may alleviate the poor growth performance caused by MG infection in broilers by improving amino acid metabolism.

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Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P [Ribosome Profiling]

Project description:Translation elongation factor P (EF-P) alleviates ribosome pausing at a subset of motifs encoding consecutive proline residues and is required for growth in many organisms. Here we show that Bacillus subtilis EF-P also alleviated ribosome pausing at sequences encoding tandem prolines, and ribosomes paused within several essential genes without a corresponding growth defect in an efp mutant. The B. subtilis efp mutant is instead impaired for flagellar biosynthesis which results in the abrogation of a form of motility called swarming. We isolate swarming suppressors of efp and identify mutations in 8 genes that suppressed the efp mutant swarming defect, many of which encode conserved ribosomal proteins or ribosome-associated factors. One mutation abolished a translational pause site within the flagellar C-ring component FliY to increase flagellar number and restore swarming motility in the absence of EF-P. Our data support a model wherein EF-P-alleviation of ribosome pausing may be particularly important for macromolecular assemblies like the flagellum that require precise protein stoichiometries.

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