Structural and Enzymatic Characterization of a cAMP-Dependent Diguanylate Cyclase from Pathogenic Leptospira Species.

Leptospira interrogans serovar Copenhageni is a human pathogen that causes leptospirosis, a worldwide zoonosis. The L. interrogans genome codes for a wide array of potential diguanylate cyclase (DGC) enzymes with characteristic GGDEF domains capable of synthesizing the cyclic dinucleotide c-di-GMP, known to regulate transitions between different cellular behavioral states in bacteria. Among such enzymes, LIC13137 (Lcd1), which has an N-terminal cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA (GAF) domain and a C-terminal GGDEF domain, is notable for having close orthologs present only in pathogenic Leptospira species. Although the function and structure of GGDEF and GAF domains have been studied extensively separately, little is known about enzymes with the GAF-GGDEF architecture. In this report, we address the question of how the GAF domain regulates the DGC activity of Lcd1. The full-length Lcd1 and its GAF domain form dimers in solution. The GAF domain binds specifically cAMP (KD of 0.24µM) and has an important role in the regulation of the DGC activity of the GGDEF domain. Lcd1 DGC activity is negligible in the absence of cAMP and is significantly enhanced in its presence (specific activity of 0.13s-1). The crystal structure of the Lcd1 GAF domain in complex with cAMP provides valuable insights toward explaining its specificity for cAMP and pointing to possible mechanisms by which this cyclic nucleotide regulates the assembly of an active DGC enzyme.

Chromobacterium violaceum adaptation to low-phosphate conditions.

Chromobacterium violaceum is a free-living bacterium that inhabits low-nutrient environments such as the Amazon basin. Bacteria respond to phosphate (Pi) shortage by expressing a range of genes involved in Pi uptake and assimilation, known as the PHO regulon. Several PHO regulon genes have been annotated in the genome of C. violaceum. Here we show that C. violaceum is extremely well adapted to low-Pi conditions. Remarkably, this bacterium is able to grow in media containing only traces of Pi. The PHO regulon genes are induced upon Pi depletion, but the bacteria continued to grow under these conditions. Unlike other Proteobacteria hitherto analyzed, neither PstS nor PhoU play a role in the repression of the PHO regulon under Pi excess.

Quantifying NAD(P)H production in the upper Entner-Doudoroff pathway from Pseudomonas putida KT2440.

Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner-Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.