From a transcriptional regulator to cell division: insights into the mechanisms regulating bacterial growth, morphogenesis, cell cycle and stress response
Embargo until
2021-08-01
Date
2019-05-30
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Johns Hopkins University
Abstract
Bacterial growth and division requires regulated synthesis of macromolecules used to expand and replicate components of the cell. The conserved transcriptional regulator CdnL associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. We addressed CdnL function in the model alphaproteobacterium Caulobacter crescentus and found that cells lacking CdnL have severe morphological, growth, and cell cycle defects. Specifically, ∆cdnL cells grow slowly, have a shorter swarmer (G1) phase and are wider, more curved, and have shorter stalks compared to wild-type (WT) cells. We were able to explain several aspects of the ∆cdnL phenotype using several ‘omics data. RNA-Seq showed that ∆cdnL cells exhibited transcriptional downregulation of most major classes of biosynthetic and bioenergetics pathways as compared to WT. Consistent with this, metabolomics analysis revealed significant decreases in the levels of critical metabolites, including pyruvate, -ketoglutarate, ATP, NAD+, UDP-N-acetyl-glucosamine, and purine and pyrimidine precursors. Notably, the cell wall precursor lipid II was significantly downregulated in ∆cdnL, consistent with the morphological defects of ∆cdnL cells and with Tn-Seq data indicating that ∆cdnL is synthetic lethal with genetic perturbations that impact cell wall metabolism. ∆cdnL cells also have aberrant localization of cytoskeletal proteins MreB and CtpS which are required for maintaining proper cell shape and whose localization is dependent on availability of metabolites.
Interestingly, we find that CdnL is involved in stress response, as there is significant enrichment of the genes downregulated during carbon starvation in the set of genes downregulated in ∆cdnL. Additionally, we find that CdnL is proteolyzed in a SpoT-dependent manner during carbon or nitrogen starvation or growth in stationary phase suggesting that CdnL clearance may be required for rewiring the transcriptome to downregulate proliferative processes and promote survival during nutrient unavailability.
In addition to requiring macromolecules to duplicate components of the cell, bacterial replication necessitates accurate placement of the division machinery to produce viable progeny. We found that two conserved, small coiled-coil proteins called ZapA and ZauP are required for efficient division in Caulobacter. ZapA and ZauP colocalized at midcell. ZapA directly interacted with the essential cytoskeletal GTPase FtsZ while ZauP was recruited to midcell through its interaction with ZapA. We found that cells lacking zapA and zauP are elongated and have diffuse Z-rings. Unlike what has been reported in E. coli and B. subtilis ZapA alone or with ZauP did not affect FtsZ bundling or dynamics in vitro suggesting a bundling-independent mechanism through which ZapA and ZauP promote efficient cell division.
Through this thesis work, we have characterized molecular mechanisms by which Caulobacter cells regulate growth and division both globally – through the action of the conserved transcriptional regulator CdnL – and locally – through ZapA-ZauP-mediated effects on the cytokinetic Z-ring. Our findings shed light on the intricate mechanisms bacteria use to regulate their growth and division.
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Keywords
bacteria, caulobacter, morphogenesis, cell cycle, stress response, cell division, bacterial cell biology