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Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.

Background: Nucleotide-signaling pathways are complex systems that allow bacteria to rapidly respond to stress. Results: Cyclic diadenosine monophosphate is essential for the growth of S. aureus, and its signaling network is intricately interconnected with the stringent response. Conclusion: Cross-talk between different nucleotide-signaling networks is more common than previously anticipated. Significance: Bacteria have evolved intricate signaling networks to survive., Nucleotide-signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to external stimuli. The stringent response alarmones guanosine tetra- (ppGpp) and pentaphosphate (pppGpp) control a global response allowing cells to adapt to starvation conditions such as amino acid depletion. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that this signaling nucleotide is essential for the growth of Staphylococcus aureus, and its increased production during late growth phases indicates that c-di-AMP controls processes that are important for the survival of cells in stationary phase. By examining the transcriptional profile of cells with high levels of c-di-AMP, we reveal a significant overlap with a stringent response transcription signature. Examination of the intracellular nucleotide levels under stress conditions provides further evidence that high levels of c-di-AMP lead to an activation of the stringent response through a RelA/SpoT homologue (RSH) enzyme-dependent increase in the (p)ppGpp levels. This activation is shown to be indirect as c-di-AMP does not interact directly with the RSH protein. Our data extend this interconnection further by showing that the S. aureus c-di-AMP phosphodiesterase enzyme GdpP is inhibited in a dose-dependent manner by ppGpp, which itself is not a substrate for this enzyme. Altogether, these findings add a new layer of complexity to our understanding of nucleotide signaling in bacteria as they highlight intricate interconnections between different nucleotide-signaling networks.

11 p.-8 fig.-5 tab., The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module., This work was supported by Programa General de Conocimiento Grants PB93-0114 and PB93-0115-C02-01.