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

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.

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Bacterial cell cycle control by citrate synthase independent of enzymatic activity

10.1101/799742 ◽

2019 ◽

Author(s):

Matthieu Bergé ◽

Julian Pezzatti ◽

Víctor González-Ruiz ◽

Laurence Degeorges ◽

Serge Rudaz ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Citrate Synthase ◽

Caulobacter Crescentus ◽

Tricarboxylic Acid Cycle ◽

Cell Cycle Control ◽

Tca Cycle ◽

Cell Cycle Regulators ◽

Central Metabolism ◽

Cycle Enzyme

ABSTRACTCoordination of cell cycle progression with central metabolism is fundamental to all cell types and likely underlies differentiation into dispersal cells in bacteria. How central metabolism is monitored to regulate cell cycle functions is poorly understood. A forward genetic selection for cell cycle regulators in the polarized alpha-proteobacterium Caulobacter crescentus unearthed the uncharacterized CitA citrate synthase, a TCA (tricarboxylic acid) cycle enzyme, as unprecedented checkpoint regulator of the G1→S transition. We show that loss of the CitA protein provokes a (p)ppGpp alarmone-dependent G1-phase arrest without apparent metabolic or energy insufficiency. While S-phase entry is still conferred when CitA is rendered catalytically inactive, the paralogous CitB citrate synthase has no overt role other than sustaining TCA cycle activity when CitA is absent. With eukaryotic citrate synthase paralogs known to fulfill regulatory functions, our work extends the moonlighting paradigm to citrate synthase coordinating central (TCA) metabolism with development and perhaps antibiotic tolerance in bacteria.

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Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cells

BMC Biology ◽

10.1186/s12915-021-00955-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Léonard Hérault ◽

Mathilde Poplineau ◽

Adrien Mazuel ◽

Nadine Platet ◽

Élisabeth Remy ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Cycle Regulators ◽

Potential Mechanism ◽

Rna Seq ◽

Hematopoietic Stem ◽

Age Related ◽

Undifferentiated State ◽

Reference Map

Abstract Background Hematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale. Results Through the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state. Conclusions Our results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

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Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cell

10.1101/2020.06.17.156893 ◽

2020 ◽

Author(s):

Léonard Hérault ◽

Mathilde Poplineau ◽

Adrien Mazuel ◽

Nadine Platet ◽

Élisabeth Remy ◽

...

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Single Cell Level ◽

Cell Cycle Regulators ◽

Potential Mechanism ◽

Rna Seq ◽

Cell Level ◽

Hematopoietic Stem ◽

Undifferentiated State ◽

Reference Map

ABSTRACTHematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging the heterogeneity of mouse HSCs evolves, which contributes to the deterioration of the immune system. Here we address the transcriptional plasticity of HSC upon aging at the single-cell resolution. Through the analysis of 15,000 young and aged transcriptomes, we reveal 15 clusters of HSCs unveiling rare and specific HSC abilities that change with age. Pseudotime ordering complemented with regulon analysis showed that the consecutive differentiation states of HSC are delayed upon aging. By analysing cell cycle at the single cell level we highlight an imbalance of cell cycle regulators of very immature aged HSC that may contribute to their accumulation in an undifferentiated state.Our results therefore establish a reference map of young and old mouse HSC differentiation and reveal a potential mechanism that delay aged HSC differentiation.

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DivL Performs Critical Cell Cycle Functions in Caulobacter crescentus Independent of Kinase Activity

Journal of Bacteriology ◽

10.1128/jb.00868-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8308-8320 ◽

Cited By ~ 39

Author(s):

Sarah J. Reisinger ◽

Sarah Huntwork ◽

Patrick H. Viollier ◽

Kathleen R. Ryan

Keyword(s):

Cell Cycle ◽

Caulobacter Crescentus ◽

Essential Gene ◽

Response Regulator ◽

Phosphorylation Site ◽

Point Mutations ◽

Phosphoryl Transfer ◽

Cell Cycle Regulators ◽

Two Component Signal Transduction ◽

Signal Transduction Proteins

ABSTRACT The Caulobacter cell cycle is regulated by a network of two-component signal transduction proteins. Phosphorylation and stability of the master transcriptional regulator CtrA are controlled by the CckA-ChpT phosphorelay, and CckA activity is modulated by another response regulator, DivK. In a screen to identify suppressors of the cold-sensitive divK341 mutant, we found point mutations in the essential gene divL. DivL is similar to histidine kinases but has a tyrosine instead of a histidine at the conserved phosphorylation site (Y550). Surprisingly, we found that the ATPase domain of DivL is not essential for Caulobacter viability. We show that DivL selectively affects CtrA phosphorylation but not CtrA proteolysis, indicating that DivL acts in a pathway independent of the CckA-ChpT phosphorelay. divL can be deleted in a strain overproducing the phosphomimetic protein CtrAD51E, but unlike ΔctrA cells expressing CtrAD51E, this strain is profoundly impaired in the control of chromosome replication and cell division. Thus, DivL performs a second function in addition to promoting CtrA phosphorylation. DivL is required for bipolar DivK localization and positively regulates DivK phosphorylation. Our results show that DivL controls two key cell cycle regulators, CtrA and DivK, and that phosphoryl transfer is not DivL's essential cellular activity.

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A Homolog of the CtrA Cell Cycle Regulator Is Present and Essential in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.183.10.3204-3210.2001 ◽

2001 ◽

Vol 183 (10) ◽

pp. 3204-3210 ◽

Cited By ~ 55

Author(s):

Melanie J. Barnett ◽

Dean Y. Hung ◽

Ann Reisenauer ◽

Lucy Shapiro ◽

Sharon R. Long

Keyword(s):

Cell Cycle ◽

Sinorhizobium Meliloti ◽

Caulobacter Crescentus ◽

Essential Gene ◽

Morphological Changes ◽

Response Regulators ◽

Promoter Regions ◽

Isolation And Characterization ◽

Multiple Cell

ABSTRACT During development of the symbiotic soil bacteriumSinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several α-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions.ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.

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Regulation of bacterial cell cycle progression by redundant phosphatases

10.1101/2020.04.29.069633 ◽

2020 ◽

Author(s):

Jérôme Coppine ◽

Andreas Kaczmarczyk ◽

Kenny Petit ◽

Thomas Brochier ◽

Urs Jenal ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Caulobacter Crescentus ◽

Model Organism ◽

Replication Initiation ◽

G1 Phase ◽

Cell Cycle Regulators ◽

Cycle Progression ◽

Two Component ◽

G1 Cells

AbstractIn the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK and PleD coordinate cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK~P) and PleD (PleD~P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK~P and PleD~P in G1 cells. However, in contrast to PleC, we do not detect kinase activity with CckN. The effects of CckN inactivation are largely masked when PleC is present, but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way well before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, suggesting the existence of adaptors specifically involved in proteolytic removal of cell cycle regulators. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions.ImportanceTwo-component signal transduction systems are widely used by bacteria to sense environmental signals and respond accordingly by modulating various cellular processes, such as cell cycle progression. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to G1 phase since both proteins are timely degraded by proteolysis just before the G1-S transition. This degradation requires new proteolytic adaptors as well as an unsuspected N-terminal motif for CckN. Our work illustrates a typical example of redundant functions between two-component proteins.

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