Our goal is to define how a bacterial cell integrates the timing of DNA replication and the polar positioning of the chromosomal origin region to control the temporal and spatial expression of cell cycle events. A lynch-pin in the temporal coordination of replication initiation and the transcription of genes encoding global cell cycle regulators is DnaA. This protein functions both to initiate replication and to activate the transcription of over 40 cell cycle-controlled genes. DnaA is controlled at multiple levels, including transcriptional regulation by differential methylation of its promoter that is linked to the progression of the replication fork, and the control of its activity by the replisome-associated HdaA protein. We will determine if changes in DNA methylation state that functions to 'clock'the sequential transcription of both dnaA and ctrA controls the temporal transcription of multiple cell cycle regulated genes, including those controlled by GcrA master regulator. We will also determine if a temporally-controlled dnaA anti- sense transcript contributes to the control of dnaA expression. The dynamic spatial deployment of proteins and the origin region of the chromosome are critical factors in cell cycle control. We have shown that upon replication of the chromosomal origin, the actual DNA sequence that moves toward, and is captured by, the cell pole is parS bound to the ParB partition protein. If the ParB/parS complex is not anchored to the pole, segregation of the rest of the chromosome is impaired and the FtsZ division ring is misplaced. We have recently identified PopZ as a protein that forms a polar polymeric network and functions to anchor ParB/parS to the new cell pole. Important questions are how the polar ribosome free zone formed by the PopZ network is established and how it functions. To define how ParB/parS moves across the cell to be captured by PopZ, we have initiated an analysis of the essential ParA segregation protein. We have generated multiple mutants of ParA and visualized ParA behavior both in vivo and in vitro. We will now identify and characterize the factors that mediate its role in DNA movement and determine the mechanism that drives chromosome segregation.
Based on our elucidation of the genetic circuitry that runs a bacterial cell cycle, we designed a new class of boron-based antibiotics that are in phase two trials. In addition, we identified a small molecule inhibitor of the MreB bacterial actin, with the goal of using this as the basis for a new family of antibiotics.
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|Schrader, Jared M; Shapiro, Lucy (2015) Synchronization of Caulobacter crescentus for investigation of the bacterial cell cycle. J Vis Exp :|
|Zhou, Bo; Schrader, Jared M; Kalogeraki, Virginia S et al. (2015) The global regulatory architecture of transcription during the Caulobacter cell cycle. PLoS Genet 11:e1004831|
|Childers, W Seth; Xu, Qingping; Mann, Thomas H et al. (2014) Cell fate regulation governed by a repurposed bacterial histidine kinase. PLoS Biol 12:e1001979|
|Mera, Paola E; Kalogeraki, Virginia S; Shapiro, Lucy (2014) Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation. Proc Natl Acad Sci U S A 111:16100-5|
|Gonzalez, Diego; Kozdon, Jennifer B; McAdams, Harley H et al. (2014) The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach. Nucleic Acids Res 42:3720-35|
|Ptacin, Jerod L; Gahlmann, Andreas; Bowman, Grant R et al. (2014) Bacterial scaffold directs pole-specific centromere segregation. Proc Natl Acad Sci U S A 111:E2046-55|
|Schrader, Jared M; Zhou, Bo; Li, Gene-Wei et al. (2014) The coding and noncoding architecture of the Caulobacter crescentus genome. PLoS Genet 10:e1004463|
|Blair, Jimmy A; Xu, Qingping; Childers, W Seth et al. (2013) Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein. Structure 21:1590-601|
|Kozdon, Jennifer B; Melfi, Michael D; Luong, Khai et al. (2013) Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proc Natl Acad Sci U S A 110:E4658-67|
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