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Mayard, A.

Publications and source records attributed to Mayard, A..

4 recordsLinked to original sources

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↗

Lipopolysaccharide synthesis and traffic in the envelope of the pathogen Brucella abortus

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a new layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify a new class of bifunctional O-antigen ligases.

microbiology↗

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↗

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↗