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Staphylococcus aureus can cause disease through the production of toxins. Toxin production is autoinduced by the protein RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful vaccine target site, and RIP and inhibitory AIPs as therapeutic molecules to prevent and suppress S. aureus infections. Development of therapeutic strategies based on these molecules has been hindered by a lack of knowledge of the molecular mechanisms by which they activate or inhibit virulence. Here, we show that RAP specifically induces the phosphorylation of a novel 21-kDa protein, whereas RIP inhibits its phosphorylation. This protein was termed target of RAP (TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is not activated by RAP in the trap mutant strain, suggesting that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid analysis shows that TRAP is histidine-phosphorylated, suggesting that TRAP may be a sensor of RAP. AIPs upregulate the synthesis of RNAIII also in trap mutant strains, suggesting that TRAP and AIPs activate RNAIII synthesis via distinct signal transduction pathways. Furthermore, TRAP phosphorylation is down-regulated in the presence of AIP, suggesting that a network of signal transduction pathways regulate S. aureus pathogenesis.

Physarum polycephalum histones have been analysed by acrylamide gel electrophoresis. Two of the five major bands had electrophoretic mobilities identical with the mobilities of two bands from calf thymus histone. The P. polycephalum pattern is qualitatively the same at all stages of the synchronous mitotic cycle. Quantitative changes in the relative proportion and relative mobility of the very-lysine-rich histone are reported. I n particular, a dramatic increase in phosphate content of this histone occurred in late G2 phase with a peak where chromosome condensation is seen to be occurring in the phase contrast microscope. Phosphate content is low during S phase and the peaks of RNA synthesis.Thc presence of phosphorous, as 0-phospho-Lserine, has been clearly demonstrated in rat histone fraction FI and Langan [l] has isolated a phosphokinase and phosphatase specific for these histones. Phosphorylation can be stimulated by adenosine cyclic 3' : 5'-monophosphate and hormones. It has been suggested that histones and their chemical modifications have roles in the control of RNA synthesis, in DNA synthesis and in chromatin structure (for a review, see 121). The results presented here lead to the conclusions that phosphorylation of very-lysine-rich histonc is not directly linked with quantitative control of RNA synthesis during the mitotic cycle, nor with DNA synthesis, as has been previously suggested, but that it may be involved in the structural changes occurring during chromosome condensation.Physarum polycephalum has been used as a model of eukaryote chromosome structure for these studies because its naturally synchronous mitotic cycle gives very well synchronised mitoses without the need for interrupting growth or synchronising in any other way.Three criteria for a model of eukaryote chromosome structure have been proposed by Crick (unpublished communication) : the presence of a large amount of DNA, of histones in equal proportions and of heterogeneous nuclear DNA. P. polycephalum contains an appropriate amount of DNA, 1 pg per diploid nucleus [4] with a complexity typical of higher organisms [5,6]. Histones are present in the usual proportions [6-81. The presence of heterogeneous nuclear RNA has not been unequivocally demon-I)* strated due to practical difficulties although a highmolecular-weight ribosomal RNA precursor is present [9,10]. An additional important factor is the ability to carry out genetic analysis [3] and this is being undertaken with P. polycephalum [ll, 121 (and Haugli, unpublished results).I n the mitotic cycle of P. polycephalum DNA synthesis ( S phase) immediately follows mitosis and takes 3 to 4 h. The subsequent G2 phase takes about 5 h. RNA synthesis occurs during the S phase and in most of G2 phase with minimum values a t mitosis and the middle of interphase [4] (Fig. 8). Histone synthesis occurs during S phase [13-151. MATERIALS AND METHODS Preparation of HistonesPhysarum polycephalum was maintained in submerged shaken cultures [ 161. Synchronous surface plasmodia were grown on filter p...

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X) 4 D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the k cat for hydrolysis by a factor of 10 5 , suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32 P-labeled protein could be trapped when the enzyme was incubated with 32 Plabeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

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A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX 4 DX 6 G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLDlike activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 10 4 and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.

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Although [Cl؊ ] i regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe 3؉ -Sepharose) and anion exchange (PO-ROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca We have previously shown that [Cl Ϫ ]/[anions]/ATP/GTP differentially determine the profile of phosphorylation of a number of apical membrane proteins in airway epithelia (3, 4). We identified two of the phosphoproteins, a 19/21-kDa doublet that showed chloride concentration-dependent phosphorylation, as two isoforms of nucleoside-diphosphate kinase (3, 5). We also observed that phosphorylation of a 37-kDa protein (p37) from both human nasal and sheep tracheal epithelia was differentially modulated by nucleotides (GTP, ATP, and GDP), [Cl Ϫ ], and other anion species (3, 4). We found that GTP was the principal phosphate donor for p37 from both species; and in the presence of ATP/GDP, phosphorylation of p37 was enhanced 20-fold. However, this phosphorylation could not be inhibited by known serine/threonine and tyrosine protein kinase inhibitors, suggesting that phosphorylation was not due to the activity of these enzymes.In this study, our objective was to establish the identity of the ion-sensitive phosphoprotein p37 and to characterize its phosphorylated amino acid residue. By selectively enhancing phosphorylation of p37 (3) and by applying metal affinity chromatography, we show that p37 is identical to annexin I and provide evidence for novel annexin phosphorylation on histidine residues. Our data suggest that annexin is a component of an intracellular signaling system involving histidine phosphorylation, which is regulated by chloride concentration. EXPERIMENTAL PROCEDURESSample Preparation-A membrane fraction was prepared from ovine airway epithelia as described previously (3). Briefly, tracheal epithelia were scraped and dislodged into homogenization buffer (250 mM sucrose, 10 mM triethanolamine; one protease inhibitor tablet from Roche Molecular Biochemicals per 50 ml buffer). Pooled scrapings were homogenized and spun at 600 ϫ g for 15 min. The post-nuclear supernatant was re-spun at 100,000 ϫ g for 2 h. The pellet was resuspended in homogenization buffer and spun for 30 min at 16,000 ϫ g (this procedure was repeated three times). All procedures were conducted at 4°C. Use of lactate dehydrogenase as a cytosol marker showed that the pellet contained no cytosol (3). Aliquots of the cytosol and membrane pellet were stored in liquid nitrogen.Phosphorylation and Electronic Autoradiography-Phosphorylation and detection of phosphoproteins were performed as described previously (3). Briefl...