Protein 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.
Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae, a ...total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E. coli. After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.
Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of
encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly ...of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.
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.
•Metabolic labeling allows dSTORM analysis of pneumococcal peptidoglycan synthesis•Septal and peripheral peptidoglycan syntheses progress separately•Septal peptidoglycan is synthesized and remodeled from the start of the cell cycle•Peripheral synthesis persists after septal synthesis is completed
Synthesis of the cell wall ensures proper bacterial shape and integrity. Trouve et al. use a combination of cell labeling, single-molecule localization microscopy, and modeling to provide the first nanoscale description of cell wall assembly in Streptococcus pneumoniae, bringing new insights for bacterial morphogenesis.
The transformation of life courses in industrialized countries since the mid-twentieth century can be analyzed through the lens of life course complexity, a function of the number of transitions or ...states experienced by individuals over a given time span. Life course complexity is often measured with composite indices in a static sequence analysis framework (i.e. over a single age interval), but this method has seldom been evaluated. This paper fills this gap. We review nine indicators of life course complexity and explore the advantages of a dynamic approach to sequence analysis (i.e. examining many nested or consecutive age intervals). An application to data on the partnership histories of American and French women is used to show the properties of each measure. We conclude that simple indicators, used alone or in combination, provide a more easily interpretable description of changes and differentials in life course complexity than commonly used composite indices. In addition, we show that, for all indicators, a dynamic approach allows a more nuanced illustration of age-related transformations of life course complexity than the static approach does.
Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data ...of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients’ lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.
Separation of parent homopolymers, polystyrene and poly(ethylene oxide), from the triblock copolymer polystyrene-b-poly(ethylene oxide)-b-polystyrene was investigated by means of liquid ...chromatography techniques. Overall suitability was evaluated and compared for size exclusion chromatography, (SEC), liquid chromatography under critical conditions of enthalpic interactions (LC CC), and liquid chromatography under limiting conditions of desorption (LC LCD). Among these techniques, LC LCD was the only one able to fully separate block copolymers from both their parent homopolymers in one single run. The efficiency of the separation was proven by 1H NMR analysis of previously collected fractions.
Wasting has been associated with increased cardiovascular and all-cause mortality in chronic kidney disease (CKD). We investigated whether serum zinc-alpha2-glycoprotein (ZAG), a potent cachectic and ...lipid-mobilizing factor that is increased in patients with CKD, predicts clinical outcomes in patients on chronic hemodialysis. We quantified serum ZAG at baseline in a prospective cohort of 252 patients undergoing maintenance hemodialysis. Serum ZAG concentrations were inversely associated with serum albumin, creatinine, and triglycerides and, conversely, positively associated with age. Although ZAG is strongly linked to protein energy wasting (PEW) in patients with cancer, higher ZAG concentrations were not associated with PEW in our cohort. During a mean study follow-up of 954 days, 49 patients died and 62 patients experienced a cardiovascular event. Kaplan-Meier analysis revealed a significant correlation between serum ZAG concentrations and all-cause mortality and cardiovascular events. In separate multivariable Cox regression models, serum ZAG concentrations remained significantly associated with all-cause mortality and cardiovascular events after adjustment for demographic factors (age, sex, and dialysis vintage), metabolic parameters (serum albumin, prealbumin, triglycerides, cholesterol, normalized protein catabolic rate, and body mass index), and cardiovascular risk factors (diabetes, dyslipidemia, history of cardiovascular disease, smoking, and diuretic use as a proxy of residual renal function). Thus, serum ZAG appears to be a strong and independent predictor of mortality and cardiovascular events in patients with end-stage renal disease. Further studies are necessary to confirm this association and to elucidate the underlying mechanisms.
Crop-livestock integration is a theoretical ideal for sustainable agriculture. However, the number of European crop-livestock farms has decreased due to multiple factors (e.g. agricultural policies ...and work constraints). Crop-livestock integration beyond the farm level (e.g. through grain-manure exchanges) is a relevant option to address these limiting factors. However, this integration within farmer groups is challenging because it requires collective redesign to address organizational issues.
We developed a participatory approach that includes the serious game Dynamix to support the co-design of scenarios of crop-livestock integration among farms.
The approach consists of six steps: (1) initial group meeting to define the problem; (2) farmer interviews to identify motivations and collect technical and economic farm data; (3) scenario co-design meeting using the serious game Dynamix, including a spatially explicit board game and a simulation model; (4) multicriteria evaluation of these scenarios at the individual farm and group levels using the simulation model included in Dynamix; (5) group meeting to discuss the results; and (6) monitoring of selected scenario implementation. We applied this methodology with two groups of farmers in southwestern France.
In the two groups, crop farmers wanted to diversify their cropping systems and use manure to improve soil quality. Livestock farmers were interested in local and non-GMO feed for their animals. The scenario they selected included i) inserting cereal-legume mixtures into crop rotations on crop farms to be sold to livestock farms and ii) transferring manure from livestock farms to crop farms. In this scenario, the predicted overall gross margin increased more for livestock farmers (median = 29.90 €/ha) than for crop farmers (median = 6.60 €/ha). Nitrogen balance management was improved: crop farmers decreased their use of mineral fertilizer by 2.8–17.4 kg/ha/year; livestock farmers decreased their feed inputs improving local feed self-sufficiency. However, farmers' workload and management complexity increased, with 22–54 h of additional work per farmer per year. Compared to other scenarios, trade-offs between individual farm and group benefits resulted in greater local autonomy in inputs but lower autonomy in decision-making. In the two groups, discussions improved trust, which is a key ingredient for transitioning to integration beyond the farm level.
Our study is the first to use a standardized participatory approach based on a serious game to support the complex issue of crop-livestock integration beyond the farm level. Applying the approach to a case-study revealed its strong potential. It can easily be scaled-out to other agricultural contexts.
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•The approach co-designs crop-livestock integration among farms, as a relevant option for limiting inputs and pollution.•The serious game Dynamix allows a spatially explicit redesign at individual farm and group level.•Scenarios include an explicit design of the logistics of integration and multicriteria evaluation at farm and group level.•The scenario selected reduces logistical, legal and trust barriers involved in integration among farms.•The serious game was applied to a case-study but can easily be scaled-out to other agricultural contexts.
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