Exploring the diversity of protein modifications: special bacterial phosphorylation systems

Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one that has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine -and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in bacterial physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein modification and homeostasis in all cellular life

The Tyrosine-Autokinase UbK Is Required for Proper Cell Growth and Cell Morphology of Streptococcus pneumoniae

International audienceProtein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.Agencia Nacional de Investigación e InnovaciónECOS-Sud France-Uruguay U20B02FOCEM-COF 03/11Agence Nationale de la Recherche ANR-18-CE11-0017/ANR-21-CE11-000

Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae

Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis

Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

BACKGROUND:In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. METHODOLOGY/PRINCIPAL FINDINGS:Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. CONCLUSIONS/SIGNIFICANCE:Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology

Molecular basis of the final step of cell division in Streptococcus pneumoniae

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.We thank the staff from the ALBA synchrotron facilities for their help during crystallographic data collection. We gratefully thank Pedro Garcia (CIB, Madrid, Spain) for providing us with the plasmid allowing overproduction of GFP-LytB. This work was supported by grants from the CNRS, the University of Lyon, the Agence National de la Recherche (ANR-18-CE11-0017-02 and ANR-19-CE15-0011-01), and the Bettencourt Schueller Foundation to C.G. The work in Spain was supported by grants BFU2017-90030-P and PID2020-115331GB-100 to J.A.H., funded by MCIN/AEI/10.13039/501100011033. The work in the United States was supported by a grant from the National Institutes of Health (GM131685). J.A.H. and C.G. supervised this work and share last authorship.Peer reviewe

Rôle des tyrosine-kinases d Escherichia coli dans la biosynthèse de l acide colanique et dans la résistance aux antibiotiques de type polymyxine

Récemment, une nouvelle famille d enzymes bactériennes capables de phosphoryler les protéines sur la tyrosine a été identifiée : les BY-kinases. Cependant, le rôle physiologique de ces enzymes est encore peu compris. Dans la première partie de ce travail, nous avons montré que la phosphorylation et la déphosphorylation cyclique de la BY-kinase Wzcca d Escherichia coli contrôle non seulement la quantité mais également la taille d un polymère exopolysaccharidique capsulaire particulier, l acide colanique. Nous avons ensuite mis en évidence le rôle essentiel de la seconde BY-kinase d E. coli, la protéine Etk, dans la résistance à la polymyxine. Enfin, nous avons identifié le site de phosphorylation d un substrat des BY-kinases, l UDP-glucose déshydrogénase, la protéine Ugd. Nous avons également démontré que cette modification, catalysée soit par la BY-kinase Wzcca soit par la BY-kinase Etk, influence la production d acide colanique et la résistance à la polymyxine. La résistance aux antibiotiques et la production de polysaccharides confèrent un avantage évident à une bactérie pathogène lors de l infection d un organisme. Par conséquent, ce travail permet de formuler l hypothèse selon laquelle les BY-kinases et leurs substrats seraient impliqués dans la régulation du pouvoir virulent de certaines bactéries pathogènes. De ce fait, ces molécules représenteraient des cibles thérapeutiques intéressantes.A new class of bacterial enzymes, named BY-kinases, have been shown to catalyze protein-tyrosine phosphorylation. Evidence of their involvement in several physiological processes has been provided, but their precise functions remain largely unknown. First, we have demonstrated that the synthesis of the exopolysaccharide colanic acid in E. coli cells, as regulated by the activity of the BY-kinase Wzcca, requires both the phosphorylated and the non-phosphorylated forms of this enzyme. Then, we have shown that polymyxin resistance of E. coli deficient for the expression of the other BY-kinase of E. coli, the protein Etk, is drastically altered. Finally, we have characterized the phosphorylation site of an UDP-glucose dehydrogenase, named Ugd. Moreover, we have provided in vivo evidence that this modification, catalysed either by Wzcca or by Etk, specifically controls the biosynthesis of colanic acid and polymyxine resistance. Antibiotics resistance and polysaccharides production confer a clear advantage for pathogenic bacteria during infection. Consequently, we assume that BY-kinases and their substrates would not only be involved in the regulation virulence of certain pathogenic bacteria, but would also represent interesting new therapeutic targetsLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Recent progress in our understanding of peptidoglycan assembly in Firmicutes

International audienc