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

PrkC-mediated Phosphorylation of Overexpressed YvcK Protein Regulates PBP1 Protein Localization in Bacillus subtilis mreB Mutant Cells

International audienc

Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules

AbstractThe mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands

Phosphorylation of CpgA protein enhances both its GTPase activity and its affinity for ribosome and is crucial for Bacillus subtilis growth and morphology.

International audienceIn Bacillus subtilis, the ribosome-associated GTPase CpgA is crucial for growth and proper morphology and was shown to be phosphorylated in vitro by the Ser/Thr protein kinase PrkC. To further understand the function of the Escherichia coli RsgA ortholog, CpgA, we first demonstrated that its GTPase activity is stimulated by its association with the 30 S ribosomal subunit. Then the role of CpgA phosphorylation was analyzed. A single phosphorylated residue, threonine 166, was identified by mass spectrometry. Phosphoablative replacement of this residue in CpgA induces a decrease of both its affinity for the 30 S ribosomal subunit and its GTPase activity, whereas a phosphomimetic replacement has opposite effects. Furthermore, cells expressing a nonphosphorylatable CpgA protein present the morphological and growth defects similar to those of a cpgA-deleted strain. Altogether, our results suggest that CpgA phosphorylation on Thr-166 could modulate its ribosome-induced GTPase activity. Given the role of PrkC in B. subtilis spore germination, we propose that CpgA phosphorylation is a key regulatory process that is essential for B. subtilis development

Experimental study of a nanoscale translocation ratchet

International audienc

Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae.

International audienceEukaryotic-like serine/threonine-kinases are involved in the regulation of a variety of physiological processes in bacteria. In Streptococcus pneumoniae, deletion of the single serine/threonine-kinase gene stkP results in an aberrant cell morphology suggesting that StkP participates in pneumococcus cell division. To understand the function of StkP, we have engineered various pneumococcus strains expressing truncated or kinase-dead forms of StkP. We show that StkP kinase activity, but also its extracellular and cytoplasmic domains per se, are required for pneumococcus cell division. Indeed, we observe that mutant cells show round or elongated shapes with non-functional septa and a chain phenotype, delocalized sites of peptidoglycan synthesis and diffused membrane StkP localization. To gain understanding of the underlying StkP-mediated regulatory mechanism, we show that StkP specifically phosphorylates in vivo the cell division protein DivIVA on threonine 201. Pneumococcus cells expressing non-phosphorylatable DivIVA-T201A possess an elongated shape with a polar bulge and aberrant spatial organization of nascent peptidoglycan. This brings the first evidence of the importance of StkP in relationship to the phosphorylation of one of its substrates in cell division. It is concluded that StkP is a multifunctional protein that plays crucial functions in pneumococcus cell shape and division

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae

International audienc

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae

International audienc

Comparative analysis of the Tyr-kinases CapB1 and CapB2 fused to their cognate modulators CapA1 and CapA2 from Staphylococcus aureus.

A particular class of tyrosine-kinases sharing no structural similarity with eukaryotic tyrosine-kinases has been evidenced in a large array of bacterial species. These bacterial tyrosine-kinases are able to autophosphorylate on a C-terminal tyrosine-rich motif. Their autophosphorylation has been shown to play a crucial role in the biosynthesis or export of capsular polysaccharide. The analysis of the first crystal structure of the staphylococcal tyrosine kinase CapB2 associated with the activating domain of the transmembrane modulator CapA1 had brought conclusive explanation for both the autophosphorylation and activation processes. In order to explain why CapA1 activates CapB2 more efficiently than its cognate transmembrane modulator CapA2, we solved the crystal structure of CapA2B2 and compared it with the previously published structure of CapA1B2. This structural analysis did not provide the expected clues about the activation discrepancy observed between the two modulators. Staphylococcus aureus also encodes for a CapB2 homologue named CapB1 displaying more than 70% sequence similarity and being surprisingly nearly unable to autophosphorylate. We solved the crystal structure of CapA1B1 and carefully compare it with the structure of CapA1B2. The active sites of both proteins are highly conserved and the biochemical characterization of mutant proteins engineered to test the importance of small structural discrepancies identified between the two structures did not explain the inactivity of CapB1. We thus tested if CapB1 could phosphorylate other protein substrates or hydrolyze ATP. However, no activity could be detected in our in vitro assays. Taken together, these data question about the biological role of the homologous protein pairs CapA1/CapB1 and CapA2/CapB2 and we discuss about several possible interpretations

Pseudodicentric Chromosome Originating from an X-Autosome Translocation in a Male Patient with Cryptozoospermia

International audienceGenetic factors are responsible for 15% of male infertility conditions. Numerical and structural chromosomal anomalies are validated genetic factors leading to spermatogenic quantitative defects, with a frequency depending on the severity of the phenotype. Among the structural chromosomal rearrangements, dicentric chromosomes are generally observed in robertsonian translocations or in cases of Y chromosome isodicentrics. In X-autosome translocations, male carriers are generally infertile, regardless of the position of the breakpoint, due to interrupted spermatogenesis. We report an infertile man bearing an unusual balanced (X;22) translocation, with a centromeric X breakpoint generating a derivative pseudodicentric chromosome psu dic(22;X). Extensive cytogenetic analyses were necessary to determine the precise nature of the derivative chromosome. The likely cause of the reproductive phenotype of the patient is discussed based on meiotic chromosomal conformation