ABSTRACT
Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter. IMPORTANCE: The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.
Subject(s)
Bacterial Proteins , Caulobacter crescentus , Gene Expression Regulation, Bacterial , Protease La , Proteolysis , Signal Transduction , Caulobacter crescentus/genetics , Caulobacter crescentus/metabolism , Caulobacter crescentus/enzymology , Caulobacter crescentus/growth & development , Protease La/metabolism , Protease La/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Heme/metabolism , Histidine KinaseABSTRACT
Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in B. abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.
Subject(s)
Bacterial Proteins , Brucella abortus , Caulobacter crescentus , DNA Methylation , Gene Expression Regulation, Bacterial , Nanopore Sequencing , Brucella abortus/genetics , Brucella abortus/metabolism , Caulobacter crescentus/genetics , Caulobacter crescentus/metabolism , Nanopore Sequencing/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)ABSTRACT
In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation in Caulobacter 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. In Caulobacter, 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.
ABSTRACT
Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and show excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in Brucella abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.
ABSTRACT
Every protein progresses through a natural lifecycle from birth to maturation to death; this process is coordinated by the protein homeostasis system. Environmental or physiological conditions trigger pathways that maintain the homeostasis of the proteome. An open question is how these pathways are modulated to respond to the many stresses that an organism encounters during its lifetime. To address this question, we tested how the fitness landscape changes in response to environmental and genetic perturbations using directed and massively parallel transposon mutagenesis in Caulobacter crescentus. We developed a general computational pipeline for the analysis of gene-by-environment interactions in transposon mutagenesis experiments. This pipeline uses a combination of general linear models (GLMs), statistical knockoffs, and a nonparametric Bayesian statistical model to identify essential genetic network components that are shared across environmental perturbations. This analysis allows us to quantify the similarity of proteotoxic environmental perturbations from the perspective of the fitness landscape. We find that essential genes vary more by genetic background than by environmental conditions, with limited overlap among mutant strains targeting different facets of the protein homeostasis system. We also identified 146 unique fitness determinants across different strains, with 19 genes common to at least two strains, showing varying resilience to proteotoxic stresses. Experiments exposing cells to a combination of genetic perturbations and dual environmental stressors show that perturbations that are quantitatively dissimilar from the perspective of the fitness landscape are likely to have a synergistic effect on the growth defect.
ABSTRACT
Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address if the degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as there is diminished expression of many FixLJ-activated genes in ∆lon. However, no physiological changes in response to microaerobic conditions occurred upon loss of Lon, suggesting that FixT dynamics are not a major contributor to fitness in oxygen limiting conditions. Similarly, stabilization of FixT in ∆lon strains does not contribute to any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during adaptation to long term aerobic growth. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter.
ABSTRACT
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 ∆lon strain was performed to explore lon's role in DNA damage survival. The ∆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 strain's 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. IMPORTANCE The 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.
Subject(s)
Caulobacter crescentus , Humans , Caulobacter crescentus/metabolism , DNA, Bacterial/metabolism , Proteomics , Proteome , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Damage , Gene Expression Regulation, BacterialABSTRACT
Bacteriophage PineapplePizza is a podovirus infecting Microbacterium foliorum NRRL B-24224. The genome is 16,662 bp long and contains 23 predicted protein-coding genes. Interestingly, PineapplePizza shows amino acid similarities to well-studied Bacillus subtilis phage phi29.
ABSTRACT
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 Δ lon strain was performed to explore lon's role in DNA damage survival. The Δ 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 strain's 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. IMPORTANCE: The 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 .
ABSTRACT
The DNA clamp loader is critical to the processivity of the DNA polymerase and coordinating synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full-length τ form and shorter γ form. These are conserved across bacterial species, and three distinct mechanisms have been found to create them: ribosomal frameshift, transcriptional slippage, and, in Caulobacter crescentus, partial proteolysis. This conservation suggests that DnaX processing is evolutionarily important, but its role remains unknown. Here we find a bias against switching from expression of a wild-type dnaX to a nonprocessable τ-only allele in Caulobacter. Despite this bias, cells are able to adapt to the τ-only allele with little effect on growth or morphology and only minor defects during DNA damage. Motivated by transposon sequencing, we find that loss of the gene sidA in the τ-only strain slows growth and increases filamentation. Even in the absence of exogenous DNA damage treatment, the ΔsidA τ-only double mutant shows induction of and dependence on recA, likely due to a defect in resolution of DNA damage or replication fork stalling. We find that some of the phenotypes of the ΔsidA τ-only mutant can be complemented by expression of γ but that an overabundance of τ-only dnaX is also detrimental. The data presented here suggest that DnaX processing is important during resolution of DNA damage events during DNA replication stress. Although the presence of DnaX τ and γ forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both τ and γ is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms are important during the resolution of DNA replication stress. IMPORTANCE Though the presence of DnaX τ and γ forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both τ and γ is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms is important during the resolution of DNA replication stress.
Subject(s)
Bacterial Proteins , Caulobacter crescentus , DNA Polymerase III , DNA Replication , Bacterial Proteins/genetics , DNA Polymerase III/genetics , Caulobacter crescentus/genetics , DNA, Bacterial/geneticsABSTRACT
Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the âº-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.
Subject(s)
Caulobacter crescentus , Caulobacter , Caulobacter/metabolism , Esterases , Cell Membrane/metabolism , Cell Wall/metabolism , Caulobacter crescentus/metabolism , Anti-Bacterial Agents , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation in Caulobacter crescentus, SpoT synthesizes the secondary messengers (p)ppGpp, which affect transcription by binding RNA polymerase to downregulate anabolic genes. (p)ppGpp also impacts expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. In Caulobacter, 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 is unclear. Here, we show that CdnL is downregulated post-translationally during starvation in a manner dependent on SpoT and the ClpXP protease. Inappropriate 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.
ABSTRACT
Anseraureola, Pondwater, and Yasuo are bacteriophages with siphovirus morphology that infect Microbacterium foliorum NRRL B-24224. They were isolated from soil collected in Amherst, Massachusetts, and have genome lengths between 17,362 bp and 17,453 bp. These phages each contain 25 predicted protein-coding genes and are assigned to phage cluster EE.
ABSTRACT
In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is easy to alter. In vitro, wild-type ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that the rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states.
Subject(s)
Escherichia coli Proteins , Protease La , ATPases Associated with Diverse Cellular Activities/metabolism , Adenosine Triphosphate/metabolism , Endopeptidase Clp/chemistry , Escherichia coli Proteins/metabolism , Hydrolysis , Protease La/metabolism , Protein Folding , Substrate SpecificityABSTRACT
Regulatory proteolysis targets properly folded clients via a combination of cis-encoded degron sequences and trans-expressed specificity factors called adaptors. SmiA of Bacillus subtilis was identified as the first adaptor protein for the Lon family of proteases, but the mechanism of SmiA-dependent proteolysis is unknown. Here, we develop a fluorescence-based assay to measure the kinetics of SmiA-dependent degradation of its client SwrA and show that SmiA-SwrA interaction and the SwrA degron were both necessary, but not sufficient, for proteolysis. Consistent with a scaffolding adaptor mechanism, we found that stoichiometric excess of SmiA caused substrate-independent inhibition of LonA-dependent turnover. Furthermore, SmiA was strictly required even when SwrA levels were high suggesting that a local increase in substrate concentration mediated by the scaffold was not sufficient for proteolysis. Moreover, SmiA function could not be substituted by thermal denaturation of the substrate, consistent with a priming adaptor mechanism. Taken together, we conclude that SmiA functions via a mechanism that is a hybrid between scaffolding and priming models.
Subject(s)
Bacillus subtilis , Bacterial Proteins , Peptide Hydrolases , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , ProteolysisABSTRACT
The understanding of bacterial gene function has been greatly enhanced by recent advancements in the deep sequencing of microbial genomes. Transposon insertion sequencing methods combines next-generation sequencing techniques with transposon mutagenesis for the exploration of the essentiality of genes under different environmental conditions. We propose a model-based method that uses regularized negative binomial regression to estimate the change in transposon insertions attributable to gene-environment changes in this genetic interaction study without transformations or uniform normalization. An empirical Bayes model for estimating the local false discovery rate combines unique and total count information to test for genes that show a statistically significant change in transposon counts. When applied to RB-TnSeq (randomized barcode transposon sequencing) and Tn-seq (transposon sequencing) libraries made in strains of Caulobacter crescentus using both total and unique count data the model was able to identify a set of conditionally beneficial or conditionally detrimental genes for each target condition that shed light on their functions and roles during various stress conditions.
Subject(s)
DNA Transposable Elements , Genes, Essential , Bayes Theorem , DNA Transposable Elements/genetics , Genes, Essential/genetics , High-Throughput Nucleotide Sequencing/methods , Mutagenesis, InsertionalABSTRACT
The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.
Subject(s)
ATP-Dependent Proteases , Caenorhabditis elegans Proteins , Caenorhabditis elegans , DNA Replication , DNA, Mitochondrial , Heteroplasmy , Mitochondria , Mitochondrial Proteins , Oxidative Phosphorylation , Transcription Factors , Animals , Humans , Animals, Genetically Modified , ATP-Dependent Proteases/genetics , ATP-Dependent Proteases/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Line , DNA Polymerase gamma/genetics , DNA Polymerase gamma/metabolism , DNA, Mitochondrial/biosynthesis , DNA, Mitochondrial/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Proteolysis , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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 image cellular (p)ppGpp. Here, we report RNA-based fluorescent sensors for the live-cell imaging of (p)ppGpp. Our sensors are 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 is now obtained under different nutritional conditions. These RNA-based sensors can be broadly adapted to study bacterial stringent response.
Subject(s)
Escherichia coli/cytology , Optical Imaging , Fluorescent Dyes , Guanosine , Guanosine Pentaphosphate , RNA , Spectrometry, FluorescenceABSTRACT
Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In Caulobacter crescentus, cell cycle-dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.