From a transcriptional regulator to cell division: insights into the mechanisms regulating bacterial growth, morphogenesis, cell cycle and stress response

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

Regulation of the transcription factor CdnL promotes adaptation to nutrient stress in <i>Caulobacter</i>

In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation inCaulobacter crescentus, SpoT synthesizes the secondary messengers guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate (collectively known as (p)ppGpp), which affect transcription by binding RNA polymerase (RNAP) to down-regulate anabolic genes. (p)ppGpp also impacts the expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. InCaulobacter, a major regulator of anabolic genes is the transcription factor CdnL. If and how CdnL is controlled during the SR and why that might be functionally important are unclear. In this study, we show that CdnL is down-regulated posttranslationally during starvation in a manner dependent on SpoT and the ClpXP protease. Artificial stabilization of CdnL during starvation causes misregulation of ribosomal and metabolic genes. Functionally, we demonstrate that the combined action of SR transcriptional regulators and CdnL clearance allows for rapid adaptation to nutrient repletion. Moreover, cells that are unable to clear CdnL during starvation are outcompeted by wild-type cells when subjected to nutrient fluctuations. We hypothesize that clearance of CdnL during the SR, in conjunction with direct binding of (p)ppGpp and DksA to RNAP, is critical for altering the transcriptome in order to permit cell survival during nutrient stress

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism