Biology subjects

Hajjar, C.

Publications and source records attributed to Hajjar, C..

3 recordsLinked to original sources

bioRxiv2026

Optimizing actin turnover in cell-like conditions

A dynamic actin cytoskeleton, characterized by rapid filament turnover, is essential for driving intracellular transport and cellular movement. Despite extensive study, the contributions of many actin-binding proteins (ABPs) that regulate the different steps of the actin turnover cycle (filament assembly, disassembly and recycling into polymerizable actin monomers) remain ill-defined. Here, we introduce novel sensitive in vitro assays to quantitatively assess how ABPs catalyze actin turnover. By accurately measuring nucleotide exchange dynamics and ATP consumption, these assays enable robust characterization of ABP activity across broad concentration ranges. Using these methods and modeling of these reactions, we systematically examined the contributions of five conserved regulators, both individually and in combination, and identified conditions that maximize turnover efficiency. We also determined that increasing F-actin concentration to cellular levels affects ABP activity. Finally, we demonstrated that rapid actin turnover is preserved during encapsulation in cell-sized vesicles using the cDICE method. Together, these advances provide versatile tools and new insights into actin cytoskeletal dynamics under physiologically relevant conditions.

cell biology↗

bioRxiv2019

Simple rules govern the diversity of bacterial nicotianamine-like metallophores

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the hosts nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis respectively. These metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or -ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH), depending on the species. Here, we explored substrate specificity of CntM by combining bioinformatics and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity was mainly due to the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity towards -ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity towards pyruvate whereas an alanine leads to the consumption of both pyruvate and -ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, opine-type metallophore diversity is mainly mediated by the absence/presence of a cntK gene encoding a histidine racemase, and the presence of an aspartate/alanine at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline.

biochemistry↗

bioRxiv2017

Pseudopaline, a staphylopine-like metallophore involved in zinc and nickel uptake in Pseudomonas aeruginosa

Metal uptake is vital for all living organisms. In metal scarce conditions, a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here, we characterize a four genes operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated to an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The metal-uptake property of the pseudopaline system appears different from that of staphylopine with a predominant effect on nickel uptake, and on zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as a zinc importer under metal scarce conditions.\n\nAUTHOR SUMMARYZinc is an essential micronutrients for bacteria, particularly important at the host-pathogen interface where the host tends to sequester metals in a so called nutritional immunity framework, and the pathogenic bacterium increases its metal uptake efforts in order to keep up with its metal requirements. Here we reveal a novel metallophore, named pseudopaline, which is synthesized and exported by Pseudomonas aeruginosa and serves for the uptake of nickel in metal poor media, and for the uptake of zinc in metal scarce conditions that mimic the chelating environment that presumably prevails within a host.

microbiology↗