Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: adaptation for different ecological niches.

Escherichia coli cyclic-AMP receptor protein (CRP) represents one of the paradigms of bacterial gene regulation. Yet despite decades of intensive study, new information continues to emerge that prompts reassessment of this classic regulatory system. Moreover, in recent years CRPs from several other bacterial species have been characterized, allowing the general applicability of the CRP paradigm to be tested. Here the properties of the E. coli, Mycobacterium tuberculosis and Pseudomonas putida CRPs are considered in the context of the ecological niches occupied by these bacteria. It appears that the cyclic-AMP-CRP regulatory system has been adapted to respond to distinct external and internal inputs across a broad sensitivity range that is, at least in part, determined by bacterial lifestyles

Metallophosphoesterases

Calcineurin-like metallophosphoesterases (MPEs) form a large superfamily of binuclear metal-ion-centre-containing enzymes that hydrolyse phosphomono-, phosphodi-or phosphotri-esters in a metal-dependent manner. The MPE domain is found in Mre11/SbcD DNA-repair enzymes, mammalian phosphoprotein phosphatases, acid sphingomyelinases, purple acid phosphatases, nucleotidases and bacterial cyclic nucleotide phosphodiesterases. Despite this functional diversity, MPEs show a remarkably similar structural fold and active-site architecture. In the present review, we summarize the available structural, biochemical and functional information on these proteins. We also describe how diversification and specialization of the core MPE fold in various MPEs is achieved by amino acid substitution in their active sites, metal ions and regulatory effects of accessory domains. Finally, we discuss emerging roles of these proteins as non-catalytic protein-interaction scaffolds. Thus we view the MPE superfamily as a set of proteins with a highly conserved structural core that allows embellishment to result in dramatic and niche-specific diversification of function

Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only similar to 30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRPMt), consistent with the relatively low dependence on cAMP of CRPMt binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis

Signalling mechanisms in Mycobacteria

The importance of inter- and intracellular signal transduction in all forms of life cannot be underestimated. A large number of genes dedicated to cellular signalling are found in almost all sequenced genomes, and Mycobacteria are no exception. What appears to be interesting in Mycobacteria is that well characterized signalling mechanisms used by bacteria, such as the histidine-aspartate phosphorelay seen in two-component systems, are found alongside signalling components that closely mimic those seen in higher eukaryotes. This review will describe the important contribution made by researchers in India towards the identification and characterization of proteins involved in two-component signalling, protein phosphorylation and cyclic nucleotide metabolism

Evolution of Complex Chemical Mixtures Reveals Combinatorial Compression and Population Synchronicity

Some of the most interesting open questions about the origins of life and molecular sciences center on chemical evolution and the spontaneous generation of complex and functional chemical species. The processes that generated the spectacular biopolymers that underlay biology demonstrate an untapped, by modern science, creative potential. We have established a robust, facile, and generally applicable platform for observing and analyzing chemical evolution using complex mixtures. While previous studies have characterized the formation of proto-polymers via chemical reactions, we systematically studied the process itself. We report empirical outcomes that were not foreseen or predicted. We have constructed an experimental platform to study the evolution of chemical systems that: (i) undergoes continuous recursive change with transitions to new chemical spaces while not converging throughout the course of the experiment, (ii) demonstrates chemical selection, during which combinatorial explosion is avoided, (iii) maintains synchronicity of molecular sub-populations, and (iv) harvests environmental energy that is stored in chemical energy. We have established some general guidelines for conducting chemical evolution. Our results suggest that chemical evolution can be adapted to produce a broad array of molecules with novel structures and functions

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability*

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning