Emergence of the Dickeya genus involved duplication of the OmpF porin and the adaptation of the EnvZ-OmpR signaling network.

Genome evolution, and more specifically gene duplication, is a key process shaping host-microorganism interaction. The conserved paralogs usually provide an advantage to the bacterium to thrive. If not, these genes become pseudogenes and disappear. Here, we show that during the emergence of the genus Dickeya, the gene encoding the porin OmpF was duplicated. Our results show that the ompF2 expression is deleterious to the virulence of Dickeya dadantii, the agent causing soft rot disease. Interestingly, ompF2 is regulated while ompF is constitutive but activated by the EnvZ-OmpR two-component system. In vitro, acidic pH triggers the system. The pH measured in four eudicotyledons increased from an initial pH of 5.5 to 7 within 8 h post-infection. Then, the pH decreased to 5.5 at 10 h post-infection and until full maceration of the plant tissue. Yet, the production of phenolic acids by the plant's defenses prevents the activation of the EnvZ-OmpR system to avoid the ompF2 expression even though environmental conditions should trigger this system. We highlight that gene duplication in a pathogen is not automatically an advantage for the infectious process and that, there was a need for our model organism to adapt its genetic regulatory networks to conserve these duplicated genes. IMPORTANCE Dickeya species cause various diseases in a wide range of crops and ornamental plants. Understanding the molecular program that allows the bacterium to colonize the plant is key to developing new pest control methods. Unlike other enterobacterial pathogens, Dickeya dadantii, the causal agent of soft rot disease, does not require the EnvZ-OmpR system for virulence. Here, we showed that during the emergence of the genus Dickeya, the gene encoding the porin OmpF was duplicated and that the expression of ompF2 was deleterious for virulence. We revealed that while the EnvZ-OmpR system was activated in vitro by acidic pH and even though the pH was acidic when the plant is colonized, this system was repressed by phenolic acid (generated by the plant's defenses). These results provide a unique- biologically relevant-perspective on the consequence of gene duplication and the adaptive nature of regulatory networks to retain the duplicated gene.

The EnvZ-OmpR Two-Component Signaling System Is Inactivated in a Mutant Devoid of Osmoregulated Periplasmic Glucans in Dickeya dadantii.

Osmoregulated periplasmic glucans (OPGs) are general constituents of alpha-, beta-, and gamma-Proteobacteria. This polymer of glucose is required for full virulence of many pathogens including Dickeya dadantii (D. dadantii). The phytopathogenic enterobacterium D. dadantii causes soft-rot disease in a wide range of plants. An OPG-defective mutant is impaired in environment sensing. We previously demonstrated that (i) fluctuation of OPG concentration controlled the activation level of the RcsCDB system, and (ii) RcsCDB along with EnvZ/OmpR controlled the mechanism of OPG succinylation. These previous data lead us to explore whether OPGs are required for other two-component systems. In this study, we demonstrate that inactivation of the EnvZ/OmpR system in an OPG-defective mutant restores full synthesis of pectinase but only partial virulence. Unlike for the RcsCDB system, the EnvZ-OmpR system is not controlled by OPG concentration but requires OPGs for proper activation.

The opgC gene is required for OPGs succinylation and is osmoregulated through RcsCDB and EnvZ/OmpR in the phytopathogen Dickeya dadantii.

Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis.

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.

The two-component system CpxAR is essential for virulence in the phytopathogen bacteria Dickeya dadantii†EC3937.

The CpxAR two-component system is present in many Proteobacteria. It controls expression of genes required to maintain envelope integrity in response to environmental injury. Consequently, this two-component system was shown to be required for virulence of several zoo-pathogens, but it has never been investigated in phyto-pathogens. In this paper, we investigate the role of the CpxAR two-component system in vitro and in vivo in Dickeya dadantii, an enterobacterial phytopathogen that causes soft-rot disease in a large variety of plant species. cpxA null mutant displays a constitutively phosphorylated CpxR phenotype as shown by direct analysis of phosphorylation of CpxR by a Phos-Tag retardation gel approach. Virulence in plants is completely abolished in cpxA or cpxR mutants of D. dadantii. In planta, CpxAR is only activated at an early stage of the infection process as shown by Phos-Tag and gene fusion analyses. To our knowledge, this is the first time that the timing of CpxAR phosphorelay activation has been investigated during the infection process by direct monitoring of response regulator phosphorylation.

Increased phosphorylation of the RcsB regulator of the RcsCDB phosphorelay in strains of Dickeya dadantii devoid of osmoregulated periplasmic glucans revealed by Phos-tag gel analysis.

Osmoregulated periplasmic glucans (OPGs) are general constituents of many proteobacteria. OPGs are important factors required for full virulence in many pathogens including Dickeya dadantii. D. dadantii causes the soft-rot disease in a wide range of plant species. The pleiotropic phenotype of opg-negative strains includes total loss of virulence and motility, and is linked to the constitutive activation of the RcsCDB phosphorelay, deduced from expression analysis of genes of the RcsCDB regulon. The constitutive activation of the RcsCDB phosphorelay in an opg-negative strain was demonstrated by direct analysis of the phosphorylation level of the RcsB regulator protein in vivo by using a Phos-tag retardation gel approach, and was correlated with the phenotype and the expression of motility genes. Data revealed a low level of RcsB phosphorylated form in the wild-type strain and a slight increase of phosphorylation in opgG mutant strains sufficient to induce the pleiotropic phenotype observed.

Inactivation of pecS restores the virulence of mutants devoid of osmoregulated periplasmic glucans in the phytopathogenic bacterium Dickeya dadantii.

Dickeya dadantii is a phytopathogenic enterobacterium that causes soft rot disease in a wide range of plant species. Maceration, an apparent symptom of the disease, is the result of the synthesis and secretion of a set of plant cell wall-degrading enzymes (PCWDEs), but many additional factors are required for full virulence. Among these, osmoregulated periplasmic glucans (OPGs) and the PecS transcriptional regulator are essential virulence factors. Several cellular functions are controlled by both OPGs and PecS. Strains devoid of OPGs display a pleiotropic phenotype including total loss of virulence, loss of motility and severe reduction in the synthesis of PCWDEs. PecS is one of the major regulators of virulence in D. dadantii, acting mainly as a repressor of various cellular functions including virulence, motility and synthesis of PCWDEs. The present study shows that inactivation of the pecS gene restored virulence in a D. dadantii strain devoid of OPGs, indicating that PecS cannot be de-repressed in strains devoid of OPGs.

Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the phosphoethanolamine transferase is encoded by opgE.

Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii.

Osmoregulated periplasmic glucans (OPGs) are general constituents of many Proteobacteria. Synthesis of these oligosaccharides is repressed by increased osmolarity of the medium. OPGs are important factors required for full virulence in many zoo- or phytopathogens including Dickeya dadantii. The phytopathogen enterobacterium D. dadantii causes soft-rot disease on a wide range of plant species. The total loss of virulence of opg-negative strains of D. dadantii is linked to the constitutive activation of the RcsCD RcsB phosphorelay highlighting relationship between this phosphorelay and OPGs. Here we show that OPGs control the RcsCD RcsB activation in a concentration-dependent manner, are required for proper activation of this phosphorelay by medium osmolarity, and a high concentration of OPGs in planta is maintained to achieve the low level of activation of the RcsCD RcsB phosphorelay required for full virulence in D. dadantii.

A novel Toxoplasma gondii nuclear factor TgNF3 is a dynamic chromatin-associated component, modulator of nucleolar architecture and parasite virulence.

In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii.

The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay.

Dickeya dadantii is a pectinolytic phytopathogen enterobacterium that causes soft rot disease on a wide range of plant species. The virulence of D. dadantii involves several factors, including the osmoregulated periplasmic glucans (OPGs) that are general constituents of the envelope of proteobacteria. In addition to the loss of virulence, opg-negative mutants display a pleiotropic phenotype, including decreased motility and increased exopolysaccharide synthesis. A nitrosoguanidine-induced mutagenesis was performed on the opgG strain, and restoration of motility was used as a screen. The phenotype of the opg mutant echoes that of the Rcs system: high level activation of the RcsCD-RcsB phosphorelay is needed to activate exopolysaccharide synthesis and to repress motility, while low level activation is required for virulence in enterobacteria. Here, we show that mutations in the RcsCDB phosphorelay system restored virulence and motility in a D. dadantii opg-negative strain, indicating a relationship between the Rcs phosphorelay and OPGs.

CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis.

The conserved prpC, prkC, cpgA locus in Bacillus subtilis encodes respectively a Ser/Thr phosphatase, the cognate sensor kinase (containing an external PASTA domain suggested to bind peptidoglycan precursors) and CpgA, a small ribosome-associated GTPase that we have shown previously is implicated in shape determination and peptidoglycan deposition. In this study, in a search for targets of PrkC and PrpC, we showed that, in vitro, CpgA itself is phosphorylated on serine and threonine, and another GTPase, the translation factor EF-Tu, is also phosphorylated by the kinase on the conserved T384 residue. Both substrates are dephosphorylated by PrpC in vitro. In addition, we identified YezB, a 10.3 kDa polypeptide, and a component of the stressosome, as a substrate for both enzymes in vitro and apparently in vivo. We propose that the PrpC/PrkC/CpgA system constitutes an important element of a regulatory network involved in the coordination of cell wall expansion and growth in B. subtilis.

The universal Kae1 protein and the associated Bud32 kinase (PRPK), a mysterious protein couple probably essential for genome maintenance in Archaea and Eukarya.

The similarities between essential molecular mechanisms in Archaea and Eukarya make it possible to discover, using comparative genomics, new fundamental mechanisms conserved between these two domains. We are studying a complex of two proteins conserved in Archaea and Eukarya whose precise biological role and biochemical function remain unknown. One of them is a universal protein known as Kae1 (kinase-asociated endopeptidase 1). The second protein is a serine/threonine kinase corresponding to the proteins Bud32 in Saccharomyces cerevisiae and PRPK (p53-related protein kinase) in humans. The genes encoding the archaeal orthologues of Kae1 and PRPK are either contiguous or even fused in many archaeal genomes. In S. cerevisiae, Kae1 and Bud32 (PRPK) belong to a chromatin-associated complex [KEOPS (kinase, endopeptidase and other proteins of small size)/EKC (endopeptidase-like kinase chromatin-associated)] that is essential for telomere elongation and transcription of essential genes. Although Kae1 is annotated as O-sialoglycoprotein endopeptidase in most genomes, we found that the Kae1 protein from Pyrococcus abyssi has no protease activity, but is an atypical DNA-binding protein with an AP (apurinic) lyase activity. The structure of the fusion protein from Methanocaldococcus jannaschii revealed that Kae1 maintains the ATP-binding site of Bud32 [corrected] in an inactive configuration. We have in fact found that Kae1 inhibits the kinase activity of Bud32 (PRPK) in vitro. Understanding the precise biochemical function and biological role of these two proteins (which are probably essential for genome maintenance) remains a major challenge.

The GTPase, CpgA(YloQ), a putative translation factor, is implicated in morphogenesis in Bacillus subtilis.

YloQ, from Bacillus subtilis, was identified previously as an essential nucleotide-binding protein of unknown function. YloQ was successfully over-expressed in Escherichia coli in soluble form. The purified protein displayed a low GTPase activity similar to that of other small bacterial GTPases such as Bex/Era. Based on the demonstrated GTPase activity and the unusual order of the yloQ G motifs, we now designate this protein as CpgA (circularly permuted GTPase). An unexpected property of this low abundance GTPase was the demonstration, using gel filtration and ultracentrifugation analysis, that the protein formed stable dimers, dependent upon the concentration of YloQ(CpgA), but independent of GTP. In order to investigate function, cpgA was placed under the control of the pspac promotor in the B. subtilis chromosome. When grown in E or Spizizen medium in the absence of IPTG, the rate of growth was significantly reduced. A large proportion of the cells exhibited a markedly perturbed morphology, with the formation of swollen, bent or 'curly' shapes. To confirm that this was specifically due to depleted CpgA a plasmid-borne cpgA under pxyl control was introduced. This restored normal cell shape and growth rate, even in the absence of IPTG, provided xylose was present. The crystal structure of CpgA(YloQ) suggests a role as a translation initiation factor and we discuss the possibility that CpgA is involved in the translation of a subset of proteins, including some required for shape maintenance.

Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5'-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides -1245 to -625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides -1929 to -1067 and -456 to -222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii.

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis.

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.

Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes.

PrkC was shown to be a eukaryotic-like (Hanks-type) protein kinase from Bacillus subtilis with a structural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF beta or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane-spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co-transcribed with prkC, is also required for normal biofilm and spore formation.