Biology subjects

Zeinert, R. D.

Publications and source records attributed to Zeinert, R. D..

5 recordsLinked to original sources

bioRxiv2023

Proteomic survey of the DNA damage response in Caulobacter crescentus reveals evidence of post-transcriptional control

The bacterial DNA damage response is a critical, coordinated response to endogenous and exogenous sources of DNA damage. Response dynamics are dependent on coordinated synthesis and loss of relevant proteins. While much is known about its global transcriptional control, changes in protein abundance that occur upon DNA damage are less well characterized at the system level. Here, we perform a proteome-wide survey of the DNA damage response in Caulobacter crescentus. We find that while most protein abundance changes upon DNA damage are readily explained by changes in transcription, there are exceptions. The survey also allowed us to identify the novel DNA damage response factor, YaaA, which has been overlooked by previously published, transcription- focused studies. A similar survey in a {Delta}lon strain was performed to explore lons role in DNA damage survival. The {Delta}lon strain had a smaller dynamic range of protein abundance changes in general upon DNA damage compared to the wild type strain. This system-wide change to the dynamics of the response may explain this strains sensitivity to DNA damage. Our proteome survey of the DNA damage response provides additional insight into the complex regulation of stress response and nominates a novel response factor that was overlooked in prior studies. IMPORTANCEThe DNA damage response helps bacteria to react to and potentially survive DNA damage. The mutagenesis induced during this stress response contributes to the development of antibiotic resistance. Understanding how bacteria coordinate their response to DNA damage could help us to combat this growing threat to human health. While the transcriptional regulation of the bacterial DNA damage response has been characterized, this study is the first to our knowledge to assess the proteomic response to DNA damage in Caulobacter.

microbiology↗

bioRxiv2021

Live-Cell Imaging of (p)ppGpp with RNA-based Fluorescent Sensors

Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to directly image and measure cellular (p)ppGpp. Here, we report a type of RNA-based fluorescent sensors for live-cell imaging of (p)ppGpp. Our sensor is engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels can now be obtained under different nutritional conditions. We predict that these RNA-based sensors will be broadly adapted to study bacterial stringent response.

cell biology↗

bioRxiv2020

Degradation of Lon in Caulobacter crescentus

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about post-translational control of these critical machines. In this study we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates such as the replication initiator DnaA to lethally low levels. Taken together, this work demonstrates a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell. ImportanceProteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

microbiology↗

bioRxiv2019

The Lon protease links nucleotide metabolism with proteotoxic stress

During stress all cells must maintain proteome quality while sustaining critical processes like DNA replication. In bacteria, the Lon protease is the central route for degradation of misfolded proteins. Here, we show that in Caulobacter crescentus Lon controls dNTP pools during stress through degradation of the transcription factor CcrM. We find that elevated dNTP/NTP ratios in {Delta}lon cells protects them from deletion of otherwise essential dTTP-producing pathways and shields them from lethality of hydroxyurea, known to catastrophically deplete dNTPs. Increased dNTP production in {Delta}lon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limiting protease and Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with the availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth or unanticipated proteotoxic stress. HighlightsO_LIdCTP deaminase (DCD) is dispensable when Lon protease is absent due to increased dNTP pools. C_LIO_LIStabilization of the Lon substrate CcrM transcriptionally upregulates ribonucleotide reductase, affording protection against hydroxyurea. C_LIO_LIMisfolded proteins can competitively inhibit CcrM degradation by the Lon protease. C_LIO_LITitration of protein quality control is a mechanism that allows cells to respond to stresses that lack dedicated signal response pathways. C_LI

microbiology↗

bioRxiv2018

A legacy role for DNA binding of Lon protects against genotoxic stress

DNA binding proteins are essential for cellular life, but persistently bound complexes have toxic consequences. Here we show that the proteotoxic responsive bacterial protease Lon clears proteins from DNA to promote genotoxic stress resistance. Purified Lon binds DNA and degrades neighboring bound proteins, while a fully active DNA-blind Lon variant does not. This variant can degrade substrates as normal during unstressed growth, complements pleotropic phenotypes of {Delta}lon, including proteotoxic resilience, but remains sensitive to genotoxic stresses and fails to degrade proteins efficiently during DNA damage. Transposon sequencing reveals that {Delta}lon is vulnerable to loss of protein-DNA eviction factors and we use dynamic nucleoid occupancy profiling to show that chromosome-wide protein turnover relies on Lon DNA binding. Finally, disrupting Lon binding to mitochondria genomes also results in genotoxic stress sensitivity, consistent with the bacterial ancestry of this organelle. We propose that clearance of persistent proteins from DNA by Lon originated in free-living -proteobacteria and maintained during the evolution of mitochondria. SummaryDNA binding by the Lon protease protects against genotoxic damage in a manner preserved from bacteria to mitochondria.

microbiology↗