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Myosins play a key role in many cellular processes such as cell migration, adhesion, intracellular trafficking and internalization processes, making them ideal targets for bacteria. Through selected examples, such as enteropathogenic E. coli (EPEC), Neisseria, Salmonella, Shigella, Listeria or Chlamydia, this review aims to illustrate how bacteria target and hijack host cell myosins in order to adhere to the cell, to enter the cell by triggering their internalization, to evade from the cytosolic autonomous cell defense, to promote the biogenesis of intracellular replicative niche, to disseminate in tissues by cell-to-cell spreading, to exit out the host cell, and also to evade from macrophage phagocytosis. It highlights the diversity and sophistication of the strategy evolved by bacteria to manipulate one of their privileged targets, the actin cytoskeleton.

Bacteria of the order Legionellales, such as Legionella pneumophila, the agent of Legionnaires' disease, and Coxiella burnetii, the agent of Q fever, are widely recognized as human pathogens. While our view of the Legionellales is often limited to clinical isolates, ecological surveys are continually uncovering new members of the Legionellales that do not fall into the recognized pathogenic species. Here we emphasize that most of these Legionellales are nonpathogenic forms that have evolved symbiotic lifestyles with nonvertebrate hosts. The diversity of nonpathogenic forms remains, however, largely underexplored. We conjecture that its characterization, once contrasted with the data on pathogenic species, will reveal novel highlights on the mechanisms underlying lifestyle transitions of intracellular bacteria, including the emergence of pathogenesis and mutualism, transmission routes, and host specificity.

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Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.

Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.

trans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Trans-translation is a ribosome rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogenLegionella pneumophilaand other related species that can also cause Legionnaires disease (LD). Consistent with the essential nature of trans-translation inL. pneumophila, KKL-35 inhibits growth of all tested strains at sub-micromolar concentrations. KKL-35 is also active against other LD-causingLegionellaspecies. KKL-35 remains equally active againstL. pneumophilamutants that have evolved resistance to macrolides. KKL-35 inhibits multiplication ofL. pneumophilain human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 is not synergistic with other ribosome-targeting antibiotics and does not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remains active againstL. pneumophilamutants expressing an alternate ribosome-rescue system and lacking tmRNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogenL. pneumophilaand with no detectable resistance. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila . Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity ( LeClaire et al., 2008 ). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits ( Vadlamudi et al., 2004 ; LeClaire et al., 2008 ; Narayanan et al., 2011 ; LeClaire et al., 2015 ). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation ( Narayanan et al., 2011 ; LeClaire et al., 2015 ). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm ( Michard et al., 2015 ). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.

International audience; Objectives: A previous study on 12 in vitro -selected azithromycin-resistant Legionella~pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia~coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L.~pneumophila . Methods: The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L.~pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. Results: We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L.~pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. Conclusions: LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L.~pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level.

International audience; Legionella are opportunistic pathogens that develop in aquatic environments where they multiply in protozoa. When infected aerosols reach the human respiratory tract they may accidentally infect the alveolar macrophages leading to a severe pneumonia called Legionnaires' disease (LD). The ability of Legionella to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Although Legionella is a large genus comprising nearly 60 species that are worldwide distributed, only about half of them have been involved in LD cases. Strikingly, the species Legionella pneumophila alone is responsible for 90% of all LD cases. The present review summarizes the molecular approaches that are used for L. pneumophila genotyping with a major focus on the contribution of whole genome sequencing (WGS) to the investigation of local L. pneumophila outbreaks and global epidemiology studies. We report the newest knowledge regarding the phylogeny and the evolution of Legionella and then focus on virulence evolution of those Legionella species that are known to have the capacity to infect humans. Finally, we discuss the evolutionary forces and adaptation mechanisms acting on the Dot/Icm system itself as well as the role of mobile genetic elements (MGE) encoding T4ASSs and of gene duplications in the evolution of Legionella and its adaptation to different hosts and lifestyles.

Scientific Reports 5: Article number: 14602; published online: 30 September 2015; updated: 03 February 2016 In the original version of this Article, Laurent Terradot was incorrectly listed as being affiliated with ‘Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom’.

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.

Bacterial tyrosine-kinases have been demonstrated to participate in the regulation of capsule polysaccharides (CPS) and exopolysaccharides (EPS) production and export. However, discrepant data have been reported on the molecular mechanism responsible for this regulation depending on the bacterial species analyzed. Special attention was previously paid to the tyrosine-kinase Wzcca of Escherichia coli K-12, which is involved in the production of the exopolysaccharide, colanic acid, and autophosphorylates by using a cooperative two-step process. In this work, we took advantage of these observations to investigate in further detail the effect of Wzcca phosphorylation on the colanic acid production. First, it is shown that expression of the phosphorylated form of Wzc prevents production of colanic acid whereas expression of the non-phosphorylated form allows biosynthesis of this exopolysaccharide. However, we provide evidence that, in the latter case, the size distribution of the colanic acid polymer is less scattered than in the case of the wild-type strain expressing both phosphorylated and non-phosphorylated forms of Wzc. It is then demonstrated that colanic acid production is not merely regulated by an on/off mechanism and that, instead, both phosphorylated and non-phosphorylated forms of Wzc are required to promote colanic acid synthesis. Moreover, a series of data suggests that besides the involvement of phosphorylated and non-phosphorylated forms of Wzc in the production of colanic acid, two particular regions of this kinase play as such an important role in the synthesis of this exopolysaccharide: a proline-rich domain located in the N-terminal part of Wzcca, and a tyrosine cluster present in the C-terminal portion of the enzyme. Furthermore, considering that polysaccharides are known to facilitate bacterial resistance to certain environmental stresses, it is shown that the resistance of E. coli to desiccation is directly connected with the phosphorylation state of Wzcca.

International audience; The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.

Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.

Legionella pneumophilais a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence ofL. pneumophilais now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle ofL. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that therepeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation ofL. pneumophila, via its T1SS, in its internalization into host cells.

Volume 79, no. 5, p. [1936–1950][1], 2011. Page 1941: [Figure 2][2] should appear as shown below. The original [Fig. 2][2] contains an error about the Coomassie blue band of GST-LegK3 ([Fig 2B][2]) that has been corrected in the revised [Fig. 2][2]. Moreover, the figure legend explains in more

Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses., IMPORTANCE Deciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style of L. pneumophila remains the principal challenge in understanding the molecular basis of Legionella virulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on the Legionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved by Legionella to counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

International audience; Dictyostelium discoideum ACAP-A is an Arf GTPase-activating protein (GAP) involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here, we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, the efficiency of fusion between post-lysosomes and the plasma membrane was reduced, resulting in the accumulation of post-lysosomes. Moreover, internalized fluid-phase markers showed extended intracellular transit times, and the transfer kinetics of phagocyted particles from lysosomes to post-lysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA(-) cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Taken together, these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfA-dependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila . Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella -containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole.

The ptp gene of Acinetobacter johnsonii was previously reported to encode a low-molecular-mass protein, Ptp, whose amino acid sequence, predicted from the theoretical analysis of the nucleotide sequence of the gene, exhibits a high degree of similarity with those of different eukaryotic and prokaryotic phosphotyrosine-protein phophatases. We have now overexpressed the ptp gene in Escherichia coli cells, purified the Ptp protein to homogeneity by a single-step chromatographic procedure, and analysed its functional properties. We have shown that Ptp can catalyse the dephosphorylation of p-nitrophenyl phosphate and phosphotyrosine, but has no effect on phosphoserine or phosphothreonine. Its activity is blocked by ammonium molybdate and sodium orthovanadate, which are strong inhibitors of phosphotyrosine-protein phosphatases, as well as by N-ethylmaleimide and iodoacetic acid. Such specificity of Ptp for phosphotyrosine has been confirmed by the observation that it can dephosphorylate endogenous proteins phosphorylated on tyrosine, but not proteins modified on either serine or threonine. In addition, Ptp has been shown to quantitatively dephosphorylate two exogenous peptides, derived respectively from leech hirudin and human gastrin, previously phosphorylated on tyrosine. Moreover, site-directed mutagenesis experiments performed on Cys11 and Arg16, which are both present in the sequence motif (H/V)C(X5)R(S/T) typical of eukaryotic phosphotyrosine-protein phosphatases, have demonstrated that each amino acid residue is essential for the catalytic activity of Ptp. Taken together, these data provide evidence that Ptp is a member of the phosphotyrosine-protein phosphatase family. Furthermore, in search for the biological function of Ptp, we have found that it can specifically dephosphorylate an endogenous protein kinase, termed Ptk, which is known to autophosphorylate at multiple tyrosine residues in the inner membrane of Acinetobacter johnsonii cells. This represents the first identification of a protein substrate for a bacterial phosphotyrosine-protein phosphatase, and therefore constitutes a possible model for analysing the role of reversible phosphorylation on tyrosine in the regulation of microbial physiology.

Dictyostelium discoideum ACAP-A is an Arf GTPase-activating protein (GAP) involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here, we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, the efficiency of fusion between post-lysosomes and the plasma membrane was reduced, resulting in the accumulation of post-lysosomes. Moreover, internalized fluid-phase markers showed extended intracellular transit times, and the transfer kinetics of phagocyted particles from lysosomes to post-lysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA(-) cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Taken together, these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfA-dependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.

International audience; Legionella pneumophila is a paradigm of highly adapted intravacuolar pathogens that acquired the rare ability to replicate within a phagocytic cell. Here, we review recent progress about the role of Type 4 secretion system effectors involved in the biogenesis of the replicative niche, the Legionella containing vacuole.

The biosynthesis of D-glutamic acid, one of the essential components of bacterial cell-wall peptidoglycan, is catalyzed by a glutamate racemase in Escherichia coli. While the other reported glutamate racemases from various (essentially gram-positive) bacterial species did not require any specific activator, the E. coli enzyme absolutely requires the presence of the peptidoglycan precursor UDP-N-acetylmuramyl-L-alanine to catalyze the interconversion of glutamic acid isomers. A comparison of the amino acid sequences of these different enzymes was made to identify amino acid residues from the E. coli enzyme that are involved in the catalysis or binding to the activator. Site-directed mutagenesis experiments are described that demonstrate the participation of cysteines 96 and 208 in the two-base reaction mechanism of the enzyme. The construction of N- or C-terminal-truncated enzymes is also described. The attractive hypothesis that the characteristic N-terminal amino acid extension (20 residues) of the E. coli enzyme could be involved in its activation by the nucleotide precursor is disproved by these experiments.

International audience; Investigation of soil amoebae in 11 cooling towers allowed us to isolate a major unknown small-sized amoeba population (SZA). However, SZA did not appear to be specific to cooling tower ecosystems since they are also a major amoeba population found in muds isolated from different points of a water treatment plant. The SSU-rDNA sequences from SZA strains did not match any known database sequences, suggesting that SZA constitutes a new amoeba taxon. We isolated and further described one of the SZA that we named Micriamoeba tesseris. The phylogenetic analyses showed that Micriamoeba tesseris belongs to the Amebozoa and branched together with genus Echinamoeba+Vermamoeba vermiformis. Phylogenetic analyses within the Micriamoeba group distinguished different subgroups of Micriamoeba strains according to their origin, i.e. cooling tower or mud. Although Micriamoeba are able to feed on viable E. coli cells, they do not uptake virulent Legionella pneumophila strains, thus enabling them to avoid infection by Legionella. Consequently, Micriamoeba is not directly involved in L. pneumophila multiplication. However, an indirect role of Micriamoeba in Legionella risk is discussed.

Legionella pneumophila is the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses.

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.

Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.

International audience; Protein PknE from Mycobacterium tuberculosis has been overproduced and purified, and its biochemical properties have been analyzed. This protein is shown to be a eukaryotic-like (Hanks'-type) protein kinase with a structural organization similar to that of membrane-bound eukaryotic sensor serine/threonine kinases. It consists of a N-terminal catalytic domain located in the cytoplasm, linked via a single transmembrane-spanning region to an extracellular C-terminal domain. The full-length enzyme, as well as the cytosolic domain alone, can autophosphorylate on serine and threonine residues. Such autokinase activity requires the presence of a lysine residue at position 45 in subdomain II, which is known to be essential also for eukaryotic kinase activity. Involvement of PknE in the transduction of external signals into the cytosol of bacteria is proposed.

Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.

Protein-tyrosine kinases regulating bacterial exopolysaccharide synthesis autophosphorylate on tyrosines located in a conserved C-terminal region. So far no other substrates have been identified for these kinases. Here we demonstrate that Bacillus subtilis YwqD not only autophosphorylates at Tyr-228, but that it also phosphorylates the two UDP-glucose dehydrogenases (UDP-glucose DHs) YwqF and TuaD at a tyrosine residue. However, phosphorylation of YwqF and TuaD occurs only in the presence of the transmembrane protein YwqC. The presumed intracellular C-terminal part of YwqC (last 50 amino acids) seems to interact with the tyrosine-kinase and to allow YwqD-catalysed phosphorylation of the two UDP-glucose DHs, which are key enzymes for the synthesis of acidic polysaccharides. However, only when phosphorylated by YwqD do the two enzymes exhibit detectable UDP-glucose DH activity. Dephosphorylation of P-Tyr-YwqF and P-Tyr-TuaD by the P-Tyr-protein phosphatase YwqE switched off their UDP-glucose DH activity. YwqE, which is encoded by the fourth gene of the B.subtilis ywqCDEF operon, also dephosphorylates P-Tyr-YwqD.

Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.

In bacteria, several proteins have been shown to autophosphorylate on tyrosine residues, but little is known on the molecular mechanism of this modification. To get more information on this matter, we have analyzed in detail the phosphorylation of a particular autokinase, protein Wzc, from Escherichia coli K12. The analysis of the hydropathic profile of this protein indicates that it is composed of two main domains: an N-terminal domain, including two transmembrane alpha-helices, and a C-terminal cytoplasmic domain. The C-terminal domain alone can undergo autophosphorylation and thus appears to harbor the protein-tyrosine kinase activity. By contrast, the N-terminal domain is not phosphorylated when incubated either alone or in the presence of the C-domain, and does not influence the extent of phosphorylation of the C-domain. The C-domain contains six different sites of phosphorylation. Among these, five are located at the C-terminal end of the molecule in the form of a tyrosine cluster (Tyr(708), Tyr(710), Tyr(711), Tyr(713), and Tyr(715)), and one site is located upstream, at Tyr(569). The Tyr(569) residue can autophosphorylate through an intramolecular process, whereas the tyrosine cluster cannot. The phosphorylation of Tyr(569) results in an increased protein kinase activity of Wzc, which can, in turn, phosphorylate the five terminal tyrosines through an intermolecular process. It is concluded that protein Wzc autophosphorylates by using a cooperative two-step mechanism that involves both intra- and interphosphorylation. This mechanism may be of biological significance in the signal transduction mediated by Wzc.In bacteria, several proteins have been shown to autophosphorylate on tyrosine residues, but little is known on the molecular mechanism of this modification. To get more information on this matter, we have analyzed in detail the phosphorylation of a particular autokinase, protein Wzc, from Escherichia coli K12. The analysis of the hydropathic profile of this protein indicates that it is composed of two main domains: an N-terminal domain, including two transmembrane alpha-helices, and a C-terminal cytoplasmic domain. The C-terminal domain alone can undergo autophosphorylation and thus appears to harbor the protein-tyrosine kinase activity. By contrast, the N-terminal domain is not phosphorylated when incubated either alone or in the presence of the C-domain, and does not influence the extent of phosphorylation of the C-domain. The C-domain contains six different sites of phosphorylation. Among these, five are located at the C-terminal end of the molecule in the form of a tyrosine cluster (Tyr(708), Tyr(710), Tyr(711), Tyr(713), and Tyr(715)), and one site is located upstream, at Tyr(569). The Tyr(569) residue can autophosphorylate through an intramolecular process, whereas the tyrosine cluster cannot. The phosphorylation of Tyr(569) results in an increased protein kinase activity of Wzc, which can, in turn, phosphorylate the five terminal tyrosines through an intermolecular process. It is concluded that protein Wzc autophosphorylates by using a cooperative two-step mechanism that involves both intra- and interphosphorylation. This mechanism may be of biological significance in the signal transduction mediated by Wzc.

Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

The biochemical properties of the autophosphorylating protein tyrosine kinase of Acinetobacter johnsonii were analyzed in vitro. The study shows that the optimal pH value for the phosphorylation reaction is approximately 7. The enzyme activity is stimulated by magnesium and, to a lesser extent, by manganese ions, whereas calcium ions have no effect. The phosphorylation process is rapid reaching a maximum in < 2 min, and the enzyme is modified at multiple sites. Interestingly, the bacterial enzyme is insensitive to a series of molecules known to affect the activity of eukaryotic protein tyrosine kinases: genistein, quercetin, tosyllysine chloromethyl ketone, and vanadate. We concluded that, even though the overall phosphorylation reaction catalyzed by the A. johnsonii enzyme is identical to that occurring in eukaryotes, this bacterial kinase exhibits a number of specific properties and therefore probably belongs to a separate group in the general family of protein tyrosine kinases.The biochemical properties of the autophosphorylating protein tyrosine kinase of Acinetobacter johnsonii were analyzed in vitro. The study shows that the optimal pH value for the phosphorylation reaction is approximately 7. The enzyme activity is stimulated by magnesium and, to a lesser extent, by manganese ions, whereas calcium ions have no effect. The phosphorylation process is rapid reaching a maximum in < 2 min, and the enzyme is modified at multiple sites. Interestingly, the bacterial enzyme is insensitive to a series of molecules known to affect the activity of eukaryotic protein tyrosine kinases: genistein, quercetin, tosyllysine chloromethyl ketone, and vanadate. We concluded that, even though the overall phosphorylation reaction catalyzed by the A. johnsonii enzyme is identical to that occurring in eukaryotes, this bacterial kinase exhibits a number of specific properties and therefore probably belongs to a separate group in the general family of protein tyrosine kinases.

International audience; The phosphorylation of proteins at tyrosine residues is known to play a key role in the control of numerous fundamental processes in animal systems. In contrast, the biological significance of protein-tyrosine phosphorylation in bacteria, which has only been recognised recently, is still unclear. Here, we have analysed the role in Escherichia coli cells of an autophosphorylating protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb, by performing knock-out experiments on the corresponding genes, wzc and wzb, and looking at the metabolic consequences induced. The results demonstrate that the phosphorylation of Wzc, as regulated by Wzb, is directly connected with the production of a particular capsular polysaccharide, colanic acid. Thus, when Wzc is phosphorylated on tyrosine, no colanic acid is synthesised by bacteria, but when dephosphorylated by Wzb, colanic acid is produced. This process is rather specific to the pair of proteins Wzc/Wzb. Indeed, a much lesser effect, if any, on colanic acid synthesis is observed when knock-out experiments are performed on another pair of genes, etk and etp, which also encode respectively a protein-tyrosine kinase, Etk, and a phosphotyrosine-protein phosphatase, Etp, in E. coli. In addition, the analysis of the phosphorylation reaction at the molecular level reveals differences between Gram-negative and Gram-positive bacteria, namely in the number of protein components required for this reaction to occur.The phosphorylation of proteins at tyrosine residues is known to play a key role in the control of numerous fundamental processes in animal systems. In contrast, the biological significance of protein-tyrosine phosphorylation in bacteria, which has only been recognised recently, is still unclear. Here, we have analysed the role in Escherichia coli cells of an autophosphorylating protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb, by performing knock-out experiments on the corresponding genes, wzc and wzb, and looking at the metabolic consequences induced. The results demonstrate that the phosphorylation of Wzc, as regulated by Wzb, is directly connected with the production of a particular capsular polysaccharide, colanic acid. Thus, when Wzc is phosphorylated on tyrosine, no colanic acid is synthesised by bacteria, but when dephosphorylated by Wzb, colanic acid is produced. This process is rather specific to the pair of proteins Wzc/Wzb. Indeed, a much lesser effect, if any, on colanic acid synthesis is observed when knock-out experiments are performed on another pair of genes, etk and etp, which also encode respectively a protein-tyrosine kinase, Etk, and a phosphotyrosine-protein phosphatase, Etp, in E. coli. In addition, the analysis of the phosphorylation reaction at the molecular level reveals differences between Gram-negative and Gram-positive bacteria, namely in the number of protein components required for this reaction to occur.

The autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii was overproduced, purified to homogeneity and assayed for ATP binding by using the nucleotide analog 5'-p-fluorosulfonylbenzoyl adenosine. The ATP binding site of this bacterial autophosphorylating protein was found to be different from that generally used by eukaryotic protein kinases. It consists of two amino acid sequences that closely resemble the Walker motifs A and B. This observation was confirmed by site-directed mutagenesis experiments which showed, in addition, that the ATP molecule bound to these motifs is effectively employed by the bacterial protein to autophosphorylate on tyrosine. It is concluded that even though the overall autophosphorylation reaction is similar in eukaryotic and prokaryotic proteins, the mechanism involved is likely different.The autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii was overproduced, purified to homogeneity and assayed for ATP binding by using the nucleotide analog 5'-p-fluorosulfonylbenzoyl adenosine. The ATP binding site of this bacterial autophosphorylating protein was found to be different from that generally used by eukaryotic protein kinases. It consists of two amino acid sequences that closely resemble the Walker motifs A and B. This observation was confirmed by site-directed mutagenesis experiments which showed, in addition, that the ATP molecule bound to these motifs is effectively employed by the bacterial protein to autophosphorylate on tyrosine. It is concluded that even though the overall autophosphorylation reaction is similar in eukaryotic and prokaryotic proteins, the mechanism involved is likely different.

International audience; Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

The protein tyrosine kinase activity of Acinetobacter calcoaceticus was analyzed in vitro through the specific phosphorylation of an endogenous protein which is modified exclusively at tyrosine residues. A strong stimulation of this activity by cyclic AMP was observed. This finding represents the first example of a protein tyrosine kinase, in prokaryotes as well as in eukaryotes, whose functioning is cyclic nucleotide-dependent.

International audience; Acinetobacter johnsonii harbors a protein tyrosine kinase activity that is able to catalyze autophosphorylation, like a number of eukaryotic tyrosine kinases. A biochemical and genetic analysis of this enzyme was performed. Maximum phosphorylation in vitro was obtained by incubating the kinase for 2 min at pH 7.0 in the presence of 5 mM magnesium chloride. In contrast to eukaryotic enzymes, no inhibitory effect of genistein and no phosphorylation of synthetic substrates such as poly (Glu80 Tyr20) or angiotensin II were observed. The analysis of the bacterial kinase by two-dimensional gel electrophoresis revealed the presence of at least five isoforms, all phosphorylated exclusively at tyrosine, which supports the concept that autophosphorylation occurs at multiple sites within the protein. The cloning and nucleotide sequencing of the gene encoding this kinase were achieved, which represents the first molecular characterization of a gene of this type in bacteria. An open reading frame of 2199 nucleotides encoding a protein of 82,373 Da was detected. The analysis of the deduced amino acid sequence suggested a possible involvement of the enzyme in cell recognition and bacterial pathogenicity. In addition, the cloning and sequencing of the region immediately upstream of the gene encoding the kinase revealed a novel open reading frame of 426 nucleotides encoding a phosphotyrosine protein phosphatase of 16,217 Da, which indicates that autophosphorylation on tyrosine is a physiologically reversible reaction.