Proteotoxic stress induces a cell-cycle arrest by stimulating Lon to degrade the replication initiator DnaA.

The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. Cells often delay replication in the face of stressful conditions, but the underlying mechanisms remain incompletely defined. Here, we demonstrate in Caulobacter crescentus that proteotoxic stress induces a cell-cycle arrest by triggering the degradation of DnaA, the conserved replication initiator. A depletion of available Hsp70 chaperone, DnaK, either through genetic manipulation or heat shock, induces synthesis of the Lon protease, which can directly degrade DnaA. Unexpectedly, we find that unfolded proteins, which accumulate following a loss of DnaK, also allosterically activate Lon to degrade DnaA, thereby ensuring a cell-cycle arrest. Our work reveals a mechanism for regulating DNA replication under adverse growth conditions. Additionally, our data indicate that unfolded proteins can actively and directly alter substrate recognition by cellular proteases.

Phosphate starvation decouples cell differentiation from DNA replication control in the dimorphic bacterium Caulobacter crescentus.

Upon nutrient depletion, bacteria stop proliferating and undergo physiological and morphological changes to ensure their survival. Yet, how these processes are coordinated in response to distinct starvation conditions is poorly understood. Here we compare the cellular responses of Caulobacter crescentus to carbon (C), nitrogen (N) and phosphorus (P) starvation conditions. We find that DNA replication initiation and abundance of the replication initiator DnaA are, under all three starvation conditions, regulated by a common mechanism involving the inhibition of DnaA translation. By contrast, cell differentiation from a motile swarmer cell to a sessile stalked cell is regulated differently under the three starvation conditions. During C and N starvation, production of the signaling molecules (p)ppGpp is required to arrest cell development in the motile swarmer stage. By contrast, our data suggest that low (p)ppGpp levels under P starvation allow P-starved swarmer cells to differentiate into sessile stalked cells. Further, we show that limited DnaA availability, and consequently absence of DNA replication initiation, is the main reason that prevents P-starved stalked cells from completing the cell cycle. Together, our findings demonstrate that C. crescentus decouples cell differentiation from DNA replication initiation under certain starvation conditions, two otherwise intimately coupled processes. We hypothesize that arresting the developmental program either as motile swarmer cells or as sessile stalked cells improves the chances of survival of C. crescentus during the different starvation conditions.

Regulation of the general stress response sigma factor σT by Lon-mediated proteolysis.

IMPORTANCE: Regulated protein degradation is a critical process in all cell types, which contributes to the precise regulation of protein amounts in response to internal and external cues. In bacteria, protein degradation is carried out by ATP-dependent proteases. Although past work revealed detailed insights into the operation principles of these proteases, there is limited knowledge about the substrate proteins that are degraded by distinct proteases and the regulatory role of proteolysis in cellular processes. This study reveals a direct role of the conserved protease Lon in regulating σT, a transcriptional regulator of the general stress response in α-proteobacteria. Our work is significant as it underscores the importance of regulated proteolysis in modulating the levels of key regulatory proteins under changing conditions.

Improving the efficacy and reliability of rTMS language mapping by increasing the stimulation frequency.

Repetitive TMS (rTMS) with a frequency of 5-10 Hz is widely used for language mapping. However, it may be accompanied by discomfort and is limited in the number and reliability of evoked language errors. We, here, systematically tested the influence of different stimulation frequencies (i.e., 10, 30, and 50 Hz) on tolerability, number, reliability, and cortical distribution of language errors aiming at improved language mapping. 15 right-handed, healthy subjects (m = 8, median age: 29 yrs) were investigated in two sessions, separated by 2-5 days. In each session, 10, 30, and 50 Hz rTMS were applied over the left hemisphere in a randomized order during a picture naming task. Overall, 30 Hz rTMS evoked significantly more errors (20 ± 12%) compared to 50 Hz (12 ± 8%; p <.01), whereas error rates were comparable between 30/50 and 10 Hz (18 ± 11%). Across all conditions, a significantly higher error rate was found in Session 1 (19 ± 13%) compared to Session 2 (13 ± 7%, p <.05). The error rate was poorly reliable between sessions for 10 (intraclass correlation coefficient, ICC = .315) and 30 Hz (ICC = .427), whereas 50 Hz showed a moderate reliability (ICC = .597). Spatial reliability of language errors was low to moderate with a tendency toward increased reliability for higher frequencies, for example, within frontal regions. Compared to 10 Hz, both, 30 and 50 Hz were rated as less painful. Taken together, our data favor the use of rTMS-protocols employing higher frequencies for evoking language errors reliably and with reduced discomfort, depending on the region of interest.

Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus.

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole-inheriting daughter cells, resulting in aggregation-free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time-lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short-lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long-lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.

An essential regulatory function of the DnaK chaperone dictates the decision between proliferation and maintenance in Caulobacter crescentus.

Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the α-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor σ32, demonstrating that the DnaK-dependent inactivation of σ32 is a growth requirement. While most mutations occurred in the rpoH gene encoding σ32, we also identified mutations affecting σ32 activity or stability in trans, providing important new insight into the regulatory mechanisms controlling σ32 activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of σ32, and a mutation leading to increased levels of the house keeping σ70 that outcompete σ32 for binding to the RNA polymerase. We demonstrate that σ32 inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has co-opted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.

Cortical Inhibition of Face and Jaw Muscle Activity and Discomfort Induced by Repetitive and Paired-Pulse TMS During an Overt Object Naming Task.

Modulatory effects of transcranial magnetic stimulation (TMS) strongly depend on the stimulation parameters. Here, we compared the immediate, task-locked inhibitory effects on speech-related muscles and the tolerability of different TMS protocols during a language production task. Repetitive TMS (rTMS) and paired-pulse TMS (PP) were applied in 13 healthy subjects over the primary motor cortex (M1) during a finger-tapping/tongue-twisting tasks. The lowest subject-specific TMS intensity leading to movement disruptions was used for TMS over left-sided speech-related areas during picture naming. Here, time-locked PP and rTMS (10/30/50 Hz; randomized sequence) were applied. Cortical silent periods (cSPs) were analyzed from electromyography obtained from various face muscles. 30 Hz- and 50 Hz-rTMS reliably evoked tongue movement disruption (ICC = 0.65) at lower rTMS intensities compared to 10 Hz-rTMS or PP. CSPs were elicited from the left hemisphere by all TMS protocols, most reliably by PP (p < 0.001). Also, cSPs with longest durations were induced by PP. Exploratory analyses of PP suggest that the trials with strongest motor inhibitory effects (presence of cSP) were associated with more articulatory naming errors, hence hinting at the utility of TMS-elicited, facial cSP for mapping of language production areas. Higher-frequency rTMS and PP evoked stronger inhibitory effects as compared to 10 Hz-rTMS during a language task, thus enabling a probably more efficient and tolerable routine for language mapping. The spatial distribution of cranial muscle cSPs implies that TMS might affect not only M1, but also distant parts of the language network.

A Kinase-Phosphatase Switch Transduces Environmental Information into a Bacterial Cell Cycle Circuit.

The bacterial cell cycle has been extensively studied under standard growth conditions. How it is modulated in response to environmental changes remains poorly understood. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus blocks cell division and grows to filamentous cells in response to stress conditions affecting the cell membrane. Our data suggest that stress switches the membrane-bound cell cycle kinase CckA to its phosphatase mode, leading to the rapid dephosphorylation, inactivation and proteolysis of the master cell cycle regulator CtrA. The clearance of CtrA results in downregulation of division and morphogenesis genes and consequently a cell division block. Upon shift to non-stress conditions, cells quickly restart cell division and return to normal cell size. Our data indicate that the temporary inhibition of cell division through the regulated inactivation of CtrA constitutes a growth advantage under stress. Taken together, our work reveals a new mechanism that allows bacteria to alter their mode of proliferation in response to environmental cues by controlling the activity of a master cell cycle transcription factor. Furthermore, our results highlight the role of a bifunctional kinase in this process that integrates the cell cycle with environmental information.

Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

ClpAP is an auxiliary protease for DnaA degradation in Caulobacter crescentus.

The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor-mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ΔclpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ΔclpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter.

Modulation of bacterial proliferation as a survival strategy.

The cell cycle is one of the most fundamental processes in biology, underlying the proliferation and growth of all living organisms. In bacteria, the cell cycle has been extensively studied since the 1950s. Most of this research has focused on cell cycle regulation in a few model bacteria, cultured under standard growth conditions. However in nature, bacteria are exposed to drastic environmental changes. Recent work shows that by modulating their own growth and proliferation bacteria can increase their survival under stressful conditions, including antibiotic treatment. Here, we review the mechanisms that allow bacteria to integrate environmental information into their cell cycle. In particular, we focus on mechanisms controlling DNA replication and cell division. We conclude this chapter by highlighting the importance of understanding bacterial cell cycle and growth control for future research as well as other disciplines.

Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium.

Spatial asymmetry is crucial to development. One mechanism for generating asymmetry involves the localized synthesis of a key regulatory protein that diffuses away from its source, forming a spatial gradient. Although gradients are prevalent in eukaryotes, at both the tissue and intracellular levels, it is unclear whether gradients of freely diffusible proteins can form within bacterial cells given their small size and the speed of diffusion. Here, we show that the bacterium Caulobacter crescentus generates a gradient of the active, phosphorylated form of the master regulator CtrA, which directly regulates DNA replication. Using a combination of mathematical modeling, single-cell microscopy, and genetic manipulation, we demonstrate that this gradient is produced by the polarly localized phosphorylation and dephosphorylation of CtrA. Our data indicate that cells robustly establish the asymmetric fates of daughter cells before cell division causes physical compartmentalization. More generally, our results demonstrate that uniform protein abundance may belie gradients and other sophisticated spatial patterns of protein activity in bacterial cells.

Nutritional control of bacterial DNA replication.

All cells must ensure precise regulation of DNA replication initiation in coordination with growth rate and in response to nutrient availability. According to a long-standing model, DNA replication initiation is tightly coupled to cell mass increase in bacteria. Despite controversies regarding this model, recent studies have provided additional support of this idea. The exact molecular mechanisms linking cell growth with DNA replication under different nutrient conditions remain elusive. However, recent studies in Caulobacter crescentus and Escherichia coli have provided insights into the regulation of DNA replication initiation in response to starvation. These mechanisms include the starvation-dependent regulation of DnaA abundance as well as mechanisms involving the small signaling molecule (p)ppGpp. In this review, we discuss these mechanisms in the context of previous findings. We highlight species-dependent similarities and differences and consider the precise growth conditions, in which the different mechanisms are active.

The heat shock protein LarA activates the Lon protease in response to proteotoxic stress.

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.

Digital participation of brain tumour patients in the assessment and treatment of communication disorders.

Introduction: Communication deficits have a severe impact on our social interactions and health-related quality of life. Subtle communication deficits are frequently overlooked or neglected in brain tumour patients, due to insufficient diagnostics. Digital tools may represent a valuable adjunct to the conventional assessment or therapy setting but might not be readily suitable for every patient. Methods: This article summarises results of three surveys on the readiness for telemedicine among (a) patients diagnosed with high-grade glioma, (b) matched controls, and (c) speech and language therapists. The respective surveys assessed the motivation for participation in telemedical assessments and supposed influencing factors, and the use potential of digital assessment and therapy technologies in daily routine, with a spotlight on brain tumour patients and the future prospects of respective telemedical interventions. Respondents included 56 high-grade glioma patients (age median: 59 years; 48% males), 73 propensity-score matched neurologically healthy controls who were instructed to imagine themselves with a severe disease, and 23 speech and language therapists (61% <35 years; all females). Results and discussion: The vast majority of the interviewed high-grade glioma (HGG) patients was open to digitisation, felt well-equipped and sufficiently skilled. The factorial analysis showed that digital offers would be of particular interest for patients in reduced general health condition (p = 0.03) and those who live far from specialised treatment services (p = 0.03). The particular motivation of these subgroups seemed to outweigh the effects of age, equipment and internet skills, which were only significant in the control cohort. The therapists' survey demonstrated a broad consensus on the need for improving the therapy access of brain tumour patients (64%) and strengthening their respective digital participation (78%), although digitisation seems to have yet hardly entered the therapists' daily practise. In summary, the combined results of the surveys call for a joint effort to enhance the prerequisites for digital participation of patients with neurogenic communication disorders, particularly in the context of heavily burdened HGG patients with limited mobility.

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

The Protein Quality Control Network in Caulobacter crescentus.

The asymmetric life cycle of Caulobacter crescentus has provided a model in which to study how protein quality control (PQC) networks interface with cell cycle and developmental processes, and how the functions of these systems change during exposure to stress. As in most bacteria, the PQC network of Caulobacter contains highly conserved ATP-dependent chaperones and proteases as well as more specialized holdases. During growth in optimal conditions, these systems support a regulated circuit of protein synthesis and degradation that drives cell differentiation and cell cycle progression. When stress conditions threaten the proteome, most components of the Caulobacter proteostasis network are upregulated and switch to survival functions that prevent, revert, and remove protein damage, while simultaneously pausing the cell cycle in order to regain protein homeostasis. The specialized physiology of Caulobacter influences how it copes with proteotoxic stress, such as in the global management of damaged proteins during recovery as well as in cell type-specific stress responses. Our mini-review highlights the discoveries that have been made in how Caulobacter utilizes its PQC network for regulating its life cycle under optimal and proteotoxic stress conditions, and discusses open research questions in this model.

The Chaperonin GroESL Facilitates Caulobacter crescentus Cell Division by Supporting the Functions of the Z-Ring Regulators FtsA and FzlA.

The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insufficient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide.IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle.

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

A nascent polypeptide sequence modulates DnaA translation elongation in response to nutrient availability.

The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes; nevertheless, the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA, we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, is critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that translation regulation by nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions.