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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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