Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.     

Multiple specificity recognition motifs enhance plant mitochondrial RNA editing in vitro

Analysis of RNA editing in plant mitochondria has at least in vitro been hampered by very low activity. Consequently, none of the trans-acting factors involved has yet been identified. We here report that in vitro RNA editing increases dramatically when additional cognate recognition motifs are introduced into the template RNA molecule. Substrate RNAs with tandemly repeated recognition elements enhance in vitro RNA editing from 2-3% to 50-80%. The stimulation is not influenced by the editing status of a respective RNA editing site, suggesting that specific recognition of a site can be independent of the edited nucleotide itself. In vivo, attachment of the editing complex may thus be analogously initiated at sequence similarities in the vicinity of bona fide editing sites. This cis-acting enhancement decreases with increasing distance between the duplicated specificity signals; a cooperative effect is detectable up to approximately 200 nucleotides. Such repeated template constructs promise to be powerful tools for the RNA affinity identification of the as yet unknown trans-factors of plant mitochondrial RNA editing.     

Phosphodiesterase activity of CvfA is required for virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.     

Characterization of the traD operon of naphthalene-catabolic plasmid NAH7: a host-range modifier in conjugative transfer

Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to 10⁵-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).     

Cellular basis of developmental plasticity observed in heterophyllous leaf formation of Ludwigia arcuata (Onagraceae)

When heterophyllous plants of Ludwigia arcuata Walt. (Onagraceae) were transferred from aerial condition to submergence, young developing leaves were matured into leaves with intermediate shape between aerial-type and submerged-type, showing spatulate shape (spoon-shaped). This change was also induced by the exposure of plants to ethylene. On the other hand, when the plants were transferred from submergence to aerial conditions, young developing leaves were matured into intermediate-type leaves with elliptic shape (spearhead shape). Anatomical analysis revealed that the formation of spatulate leaf was caused by the reduction of the number of epidermal cells aligned in the leaf transverse direction in the basal region of the leaf while the tip regions remained as before and did not respond to this treatment. During development, the ethylene-induced spatulate leaves showed that three types of alterations in epidermal cell division were involved in this process. Changes in the distribution of cell divisions in leaf lamina were detected by the first day of ethylene exposure, and changes in the orientation of cell division planes were detected by the second day. However, changes in the number of cells aligned in the leaf transverse direction were not detected by this time. Three days after ethylene exposure, frequency of cell divisions changed, and by the time changes of cell numbers aligned in the leaf transverse direction were observed. Thus, the formation of intermediate-type leaves in L. arcuata was ascribed to the alterations of cell division patterns which was induced by ethylene.     

Co-infection of respiratory bacterium with severe acute respiratory syndrome coronavirus induces an exacerbated pneumonia in mice

SARS-CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS-CoV infection in cultured cells, which suggests that SARS development may be due to elastase-mediated, enhanced SARS-CoV infection in the lungs. To explore this possibility, we examined whether co-infection of mice with SARS-CoV and Pp, a low-pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co-infected with SARS-CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33~90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co-infection with SARS-CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.