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A CRISPRi system for efficient and rapid gene knockdown in Caulobacter crescentus

ABSTRACT: CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knockdown gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus. Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically-dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter. We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in Caulobacter.

ORGANISM(S): Caulobacter vibrioides

PROVIDER: GSE139521 | GEO | 2020/01/23

REPOSITORIES: GEO

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Bypass of an epigenetic S-phase transcriptional module by convergent nutrient stress signals

Project description:The signals feeding into bacterial S-phase transcription are poorly understood. Cellular cycling in the alpha-proteobacterium Caulobacter crescentus is driven by a complex circuit of at least three transcriptional modules that direct sequential promoter firing during the G1, early and late S cell cycle phases. In alpha-proteobacteria, the transcriptional regulator GcrA and the CcrM methyltransferase epigenetically activate promoters of cell division and polarity genes that fire in S-phase. By evolving Caulobacter crescentus cells to cycle and differentiate in the absence of the GcrA/CcrM module, we discovered that phosphate deprivation and (p)ppGpp alarmone stress signals converge on S-phase transcriptional activation. The cell cycle oscillations of the CtrA protein, the transcriptional regulator CtrA that implements G1 and late S-phase transcription, are essential in our evolved mutants, but not in wild-type cells, showing that the periodicity in CtrA abundance alone can sustain cellular cycling without GcrA/CcrM. While similar nutritional sensing occurs in other alpha-proteobacteria, GcrA and CcrM are not encoded in the reduced genomes of obligate intracellular relatives. We thus propose that the nutritional stress response induced during intracellular growth obviated the need for an S-phase transcriptional regulator.

2023-11-09 | GSE135899 | GEO

Li2009- Assymetric Caulobacter cell cycle

Project description:The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine

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Project description:The Caulobacter cell cycle includes in an asymmetric cell division that is driven by a core regulatory circuit comprised of 4 transcription factors (DnaA, GcrA, CtrA, and SciP) and a DNA methyltransferase (CcrM). Using a modified global 5M-bM-^@M-^Y RACE protocol we mapped 2,726 transcriptional start sites (TSS) in the 4mb Caulobacter genome and identified 586 cell cycle-regulated TSS. The core cell cycle circuit directly controls about 55% of cell cycle-regulated TSS by integrating multiple regulatory inputs within at least 322 promoters, providing a large number of transcription profiles from a small number of regulatory factors. Here, we identified previously unknown features of the core cell cycle circuit, including antisense TSS within dnaA and ctrA, plus newly identified TSS for ctrA and ccrM. Altogether, we identified 615 antisense TSS plus 241 genes that are transcribed from multiple TSS. The multiple TSS in the same promoter region often exhibit different cell cycle activation timing, These novel features of the global transcript profile add significant insight to the system architecture of the Caulobacter cell cycle regulatory circuit. Global 5' RACE was performed to map Transcription Start Sites in the Caulobacter NA1000 genome

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The Global Landscape of Transcription Initiation During the Caulobacter Cell Cycle [timecourse]

Project description:The Caulobacter cell cycle includes in an asymmetric cell division that is driven by a core regulatory circuit comprised of 4 transcription factors (DnaA, GcrA, CtrA, and SciP) and a DNA methyltransferase (CcrM). Using a modified global 5M-bM-^@M-^Y RACE protocol we mapped 2,726 transcriptional start sites (TSS) in the 4mb Caulobacter genome and identified 586 cell cycle-regulated TSS. The core cell cycle circuit directly controls about 55% of cell cycle-regulated TSS by integrating multiple regulatory inputs within at least 322 promoters, providing a large number of transcription profiles from a small number of regulatory factors. Here, we identified previously unknown features of the core cell cycle circuit, including antisense TSS within dnaA and ctrA, plus newly identified TSS for ctrA and ccrM. Altogether, we identified 615 antisense TSS plus 241 genes that are transcribed from multiple TSS. The multiple TSS in the same promoter region often exhibit different cell cycle activation timing, These novel features of the global transcript profile add significant insight to the system architecture of the Caulobacter cell cycle regulatory circuit. Global 5' RACE was performed to measure Transcription Start Site activity at time points of the Caulobacter NA1000 cell cycle

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Transcriptional rewiring of the GcrA/CcrM bacterial epigenetic regulatory system in closely related bacteria

Project description:Transcriptional rewiring is the regulation of different targets genes by orthologous regulators in different organisms. While this phenomenon has been observed, it has not been extensively studied, particularly in core regulatory systems. Several global cell cycle regulators are conserved in the Alphaproteobacteria, providing an excellent model to study this phenomenon. First characterized in Caulobacter crescentus, GcrA and CcrM compose a DNA methylation-based regulatory system that helps coordinate the complex life cycle of this organism. These regulators are well-conserved across Alphaproteobacteria, but the extent to which their regulatory targets are conserved is not known. In this study, the regulatory targets of GcrA and CcrM were analyzed by SMRT-seq, RNA-seq, and ChIP-seq technologies in the Alphaproteobacterium Brevundimonas subvibrioides, and then compared to those of its close relative C. crescentus that inhabits the same environment. Although the regulators themselves are highly conserved, the genes they regulate are vastly different. GcrA directly regulates 204 genes in C. crescentus, and though B. subvibrioides has orthologs to 147 of those genes, only 48 genes retained GcrA binding in their promoter regions. Additionally, only 12 of those 48 genes demonstrated significant transcriptional change in a gcrA mutant, suggesting extensive transcriptional rewiring between these organisms. Similarly, out of hundreds of genes CcrM regulates in each of these organisms, only 2 genes were found in common. When multiple Alphaproteobacterial genomes were analyzed bioinformatically for potential GcrA regulatory targets, the regulation of genes involved in DNA replication and cell division was well conserved across the Caulobacterales but not outside this order. This work suggests that significant transcriptional rewiring can occur in cell cycle regulatory systems even over short evolutionary distances.

2020-02-01 | GSE138845 | GEO

Transcriptional rewiring of the GcrA/CcrM bacterial epigenetic regulatory system in closely related bacteria

2020-02-01 | GSE138844 | GEO

The bacterial cell-cycle regulator GcrA is a Ï70 co-factor that drives gene expression from a subset of methylated promoters.

Project description:Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we find that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active Ï70-dependent promoters in vivo, but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact Ï70 interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrate that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identify a regulon of ~200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than exclusively recruitment. Examination of GcrA, RNAP, Sigma70 ChIP in PYE and in PYE + rifampicin-treated for 30 min; sigma32 and sigma54 in PYE + rifampicin-treated for 30 min

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2014-10-16 | GSE57365 | GEO

The Global Landscape of Transcription Initiation During the Caulobacter Cell Cycle [timecourse]

Project description:The Caulobacter cell cycle includes in an asymmetric cell division that is driven by a core regulatory circuit comprised of 4 transcription factors (DnaA, GcrA, CtrA, and SciP) and a DNA methyltransferase (CcrM). Using a modified global 5’ RACE protocol we mapped 2,726 transcriptional start sites (TSS) in the 4mb Caulobacter genome and identified 586 cell cycle-regulated TSS. The core cell cycle circuit directly controls about 55% of cell cycle-regulated TSS by integrating multiple regulatory inputs within at least 322 promoters, providing a large number of transcription profiles from a small number of regulatory factors. Here, we identified previously unknown features of the core cell cycle circuit, including antisense TSS within dnaA and ctrA, plus newly identified TSS for ctrA and ccrM. Altogether, we identified 615 antisense TSS plus 241 genes that are transcribed from multiple TSS. The multiple TSS in the same promoter region often exhibit different cell cycle activation timing, These novel features of the global transcript profile add significant insight to the system architecture of the Caulobacter cell cycle regulatory circuit.

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The bacterial cell-cycle regulator GcrA is a σ70 co-factor that drives gene expression from a subset of methylated promoters.

Project description:Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we find that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ70-dependent promoters in vivo, but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ70 interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrate that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identify a regulon of ~200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than exclusively recruitment.

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