Biology subjects

Hallez, R.

Publications and source records attributed to Hallez, R..

8 recordsLinked to original sources

bioRxiv2025

A metabolic hierarchy directs cell cycle transition and morphogenesis

To optimize survival, cells must align differentiation and proliferation with metabolic status. Yet, how metabolic cues fine-tune cell cycle programs and morphogenesis remain unclear. Here, we show that cytosolic redox dynamics critically govern cell cycle transitions in Caulobacter crescentus. By integrating evolutionary genetics with fluxomics, we uncover temporally orchestrated shifts in core metabolic pathways that remodel cytosolic redox across the cell cycle. Early stages channel carbon flux toward unsaturated fatty-acid synthesis and a reverse-TCA to drive cytosolic oxidation-coupled G1-S transition. The later stages depend on an enhanced forward-TCA cycle that promote cytosolic reduction-driven proliferation. Strikingly, perturbing fatty-acid synthesis or reverse/forward-TCA uncouples growth from proliferation. Intriguingly, a biphasic glucose uptake program dictates the cell cycle-stage-specific metabolism. These findings reveal an unprecedented metabolic-redox circuitry coordinating morphogenesis with cell cycle progression in a carefully choreographed menage a trois.

microbiology↗

bioRxiv2025

Cross-regulation of (p)ppGpp and c-di-GMP pathways controls a cell-cycle transition

Bacteria must adapt their physiology to changing environments, a process often orchestrated by nucleotide messengers such as (p)ppGpp and cyclic di-GMP (c-di-GMP). While (p)ppGpp promotes survival under nutrient stress by suppressing growth, c-di-GMP supports sessile lifestyles and cell differentiation. In the -proteobacterium Caulobacter crescentus, these messengers have opposite effects on the cell cycle, suggesting potential cross-regulations. Here, we uncover direct molecular cross-talk between (p)ppGpp and c-di-GMP signaling pathways. Using biochemical, structural and genetic approaches, we show that (p)ppGpp binds to the diguanylate cyclase PleD and inhibits c-di-GMP synthesis by competing with GTP at the active site. This interaction prevents c-di-GMP accumulation under nutrient-limiting conditions, linking metabolic stress to inhibition of developmental progression. Our findings reveal a new paradigm for second messenger interplay, in which one messenger directly suppresses synthesis of another. This work provides insight into how bacteria integrate metabolic and developmental cues through small-molecule cross-talk to guide adaptive behavior.

microbiology↗

bioRxiv2025

Stress-Induced Iron-Sulfur Cluster Damage as a Conserved Trigger of the Stringent Response

Pathogenic bacteria rely on the stringent response to adapt to the complex and fluctuating conditions encountered within the host. However, the mechanisms by which the stringent response senses host-induced stress remain poorly understood. Here, we identify iron-sulfur (Fe-S) cluster damage as a conserved trigger of the stringent response in major Gram-negative pathogens, including Salmonella enterica, Enterobacter cloacae, and Klebsiella pneumoniae. We demonstrate that Fe-S cluster disruption--caused by oxidative stress or metal imbalance--restricts the intracellular pools of sulfur-containing and branched-chain amino acids, thereby activating the ribosome-associated (p)ppGpp synthetase RelA. Furthermore, we show that iron availability governs recovery from Fe-S cluster damage, modulating the dynamics of the stringent response. Finally, we emphasize the dual role of (p)ppGpp in transcriptional regulation, enhancing bacterial fitness during Fe-S cluster stress while simultaneously promoting virulence by upregulating the SPI-2 type III secretion system. Together, these findings uncover a conserved mechanism by which pathogenic bacteria integrate metabolic stress into adaptive gene regulation and virulence, positioning Fe-S cluster integrity as a pivotal node linking environmental sensing to transcriptional control during infection.

microbiology↗

bioRxiv2025

Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii

The (p)ppGpp-dependent stress response is required for pathogenic bacteria to survive both outside and inside the host but the mechanisms behind this survival are mostly unknown. In this study, we characterize the (p)ppGpp metabolism in the opportunistic pathogen multi-drug-resistant Acinetobacter baumannii. We show that two stressful conditions potentially encountered during infection - iron starvation and polymyxin exposure - induce (p)ppGpp production. The absence of (p)ppGpp led to multiple consequences on the physiology of A. baumannii, including an increase of surface motility, a decrease in catalase activity, a poor survival upon nutrient starvation, a rapid killing during desiccation and a strong attenuation in a Galleria mellonella model of infection. Using a motility suppressor screen, we isolated multiple independent stringent mutations in rpoB and rpoC that suppress the (p)ppGpp-dependent phenotypes. By combining the suppressor screen with deep sequencing, we isolated dozens of additional mutants, expanding the list of putative stringent RNAP mutations described so far. Furthermore, our transcriptomic data reveal that (p)ppGpp deeply impacts the transcriptional landscape of A. baumannii on solid surface to induce many stress-related genes, including catalase and hydrophilins critical for tolerance to desiccation. This work highlights the functional interplay between (p)ppGpp and RNAP in the successful survival of A. baumannii in the environment but also during infection.

microbiology↗

bioRxiv2023

Regulation of potassium homeostasis in Caulobacter crescentus

Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Nevertheless, K+ homeostasis remains poorly studied in bacteria. Here we describe the regulation of potassium uptake systems in the oligotrophic -proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by essentially using two K+ transporters to maintain potassium homeostasis, the low affinity Kup and the high affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by improving the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABC expression, suggest that the inner membrane sensor regulatory component KdpD works as a kinase in early stages of growth and as a phosphatase to regulate transition into stationary phase. Our data also show that KdpE is not only phosphorylated by KdpD but also by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome and identified the direct targets of KdpE. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability. ImportancePotassium (K+) is a key metal ion involved in many essential cellular processes. Its transport and regulation have been mainly studied in the bacterial model species Escherichia coli and Bacillus subtilis. Here we show that the oligotroph Caulobacter crescentus can support growth at lower K+ concentrations by mainly using two K+ uptake systems, the low-affinity Kup and the high-affinity Kdp. Interestingly, in the absence of Kdp, point mutations in Kup was selected to increase affinity for K+, which improved growth in K+-depleted conditions. Using genome-wide approaches, we also determined the entire set of genes required for C crescentus to survive at low K+ concentration as well as the full K+-dependent regulon. Finally, we found that the transcriptional regulation mediated by the KdpDE two-component system is unconventional since unlike E. coli, the inner membrane sensor regulatory component KdpD works rather as a phosphatase on the phosphorylated response regulator KdpE[~]P. To our knowledge, this is the first comprehensive study of K+ homeostasis in bacteria.

microbiology↗

bioRxiv2022

The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus

Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. The ChvG/ChvI (ChvGI) TCS conserved in -proteobacteria is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to regulate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and found that ChvI regulates cell envelope architecture by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. In addition, we found that ChvI phosphorylation is also activated upon antibiotic treatment with vancomycin. We also challenged chv mutants with other cell envelope related stress and found that treatment with antibiotics targeting transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon. Finally, we observed that the sensor kinase ChvG relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer to maintain cell envelope homeostasis. ImportanceThe cell envelope is the first barrier protecting cells from harsh environmental conditions, such as temperature, pH, oxidative and osmotic imbalances. It is also an obstacle for the intake of antibiotics targeting essential cellular processes. Therefore, molecular components and systems responding to cell envelope stress and maintaining cell envelope homeostasis are important targets for drug therapy. Here we show that the two-component system ChvGI, highly conserved in free-living and pathogenic -proteobacteria, is activated upon osmotic upshift and treatment with antibiotics targeting peptidoglycan synthesis to activate the transcription of multiple genes involved in cell envelope homeostasis in Caulobacter crescentus. We also show that the kinase sensor ChvG displays a dynamic localisation pattern that changes depending on osmotic imbalance. To our knowledge this is the first two-component system reported to change his cellular localisation upon environmental stress.

microbiology↗

bioRxiv2020

Regulation of bacterial cell cycle progression by redundant phosphatases

In the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK and PleD coordinate cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK~P) and PleD (PleD~P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK~P and PleD~P in G1 cells. However, in contrast to PleC, we do not detect kinase activity with CckN. The effects of CckN inactivation are largely masked when PleC is present, but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way well before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, suggesting the existence of adaptors specifically involved in proteolytic removal of cell cycle regulators. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions. ImportanceTwo-component signal transduction systems are widely used by bacteria to sense environmental signals and respond accordingly by modulating various cellular processes, such as cell cycle progression. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to G1 phase since both proteins are timely degraded by proteolysis just before the G1-S transition. This degradation requires new proteolytic adaptors as well as an unsuspected N-terminal motif for CckN. Our work illustrates a typical example of redundant functions between two-component proteins.

microbiology↗

bioRxiv2018

Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

Sensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT domains are mainly found in metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase (SD/HD) enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp SD/HD of Caulobacter crescentus. We show that SpoT requires the ACT domain to hydrolyse ppGpp in an efficient way. In addition, our in vivo and in vitro data show that the phosphorylated version of EIIANtr (EIIANtr~P) interacts directly with the ACT to inhibit the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr~P and SpoT/Rel along with the PTSNtr-dependent regulation of (p)ppGpp accumulation upon nitrogen starvation in Sinorhizobium meliloti, a plant-associated -proteobacterium. Thus, this work suggests that -proteobacteria might have inherited from a common ancestor, a PTSNtr dedicated to modulate (p)ppGpp levels.

microbiology↗