Cyclic AMP binding to a universal stress protein in Mycobacterium tuberculosis is essential for viability.

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.

Mycobacterial STAND adenylyl cyclases: The HTH domain binds DNA to form biocrystallized nucleoids.

Mycobacteria harbor a unique class of adenylyl cyclases with a complex domain organization consisting of an N-terminal putative adenylyl cyclase domain fused to a nucleotide-binding adaptor shared by apoptotic protease-activating factor-1, plant resistance proteins, and CED-4 (NB-ARC) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal helix-turn-helix (HTH) domain. The products of the rv0891c-rv0890c genes represent a split gene pair, where Rv0891c has sequence similarity to adenylyl cyclases, and Rv0890c harbors the NB-ARC-TPR-HTH domains. Rv0891c had very low adenylyl cyclase activity so it could represent a pseudoenzyme. By analyzing the genomic locus, we could express and purify Rv0890c and find that the NB-ARC domain binds ATP and ADP, but does not hydrolyze these nucleotides. Using systematic evolution of ligands by exponential enrichment (SELEX), we identified DNA sequences that bound to the HTH domain of Rv0890c. Uniquely, the HTH domain could also bind RNA. Atomic force microscopy revealed that binding of Rv0890c to DNA was sequence independent, and binding of adenine nucleotides to the protein induced the formation of higher order structures that may represent biocrystalline nucleoids. This represents the first characterization of this group of proteins and their unusual biochemical properties warrant further studies into their physiological roles in future.

A DNA Aptamer for Cyclic Adenosine Monophosphate that Shows Adaptive Recognition.

As a ubiquitous second messenger, cyclic adenosine monophosphate (cAMP) mediates diverse biological processes such as cell growth, inflammation, and metabolism. The ability to probe these pathways would be significantly enhanced if we had a DNA-based sensor for cAMP. Herein, we describe a new, 31-base long single-stranded DNA aptamer for cAMP, denoted caDNApt-1, that was isolated by in vitro selection using systemic evolution of ligands after exponential enrichment (SELEX). caDNApt-1 has an approximately threefold higher affinity for cAMP than ATP, ADP, and AMP. Using non-denaturing gel electrophoresis and fluorescence spectroscopy, we characterized the structural changes caDNApt-1 undergoes upon binding to cAMP and revealed its potential as a cAMP sensor.

A fluorescent nucleic acid nanodevice quantitatively images elevated cyclic adenosine monophosphate in membrane-bound compartments.

cAMPhor: In the presence of cAMP, cAMPhor folds into a structure that binds DFHBI (green), increasing its fluorescence, while Alexa 647 (red) functions as a normalizing dye. It can thus be used to spatially image cAMP quantitatively in membrane-bound compartments.

Tuberculous liver abscess in an immunocompetent adult male--a rare presentation.

A 20 year old young male was admitted to our hospital with complaints of pain in upper abdomen right side, anorexia and loss of weight. Ultrasonography of the upper abdomen revealed a hypoechoic area in the left lobe of liver. Entertaining the possibility of pyogenic or amoebic lesion, the patient was started on ofloxacin and metronidazole. Failing to get any response to the therapeutic intervention, ultrasound guided aspiration was undertaken. The aspirated pus did not grow any organism in pyogenic or fungal culture but showed acid fast bacilli in Z.N. stain. The treatment was shifted to four drugs ATT and there was dramatic improvement in the clinical condition. This case is being reported to emphasize that ruling out tuberculosis may avoid unnecessary delays in the initiation of specific anti-tubercular treatment. Also a greater awareness of this rare clinical condition may prevent unwarranted surgical intervention.

Synthetic, biofunctional nucleic acid-based molecular devices.

Structural DNA nanotechnology seeks to create architectures of highly precise dimensions using the physical property that short lengths of DNA behave as rigid rods and the chemical property of Watson-Crick base-pairing that acts as a specific molecular glue with which such rigid rods may be joined. Thus DNA has been used as a molecular scale construction material to make molecular devices that can be broadly classified under two categories (i) rigid scaffolds and (ii) switchable architectures. This review details the growing impact of such synthetic nucleic acid based molecular devices in biology and biotechnology. Notably, a significant trend is emerging that integrates morphology-rich nucleic acid motifs and alternative molecular glues into DNA and RNA architectures to achieve biological functionality.

The poly dA helix: a new structural motif for high performance DNA-based molecular switches.

We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA(15)) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH(+)-H(+)A base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA(15). The pH-triggered transition between the two defined helical forms of dA(15) is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA(15) represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.