Identification of a broadly conserved family of enzymes that hydrolyze (p)ppApp.

Bacteria produce a variety of nucleotide second messengers to adapt to their surroundings. Although chemically similar, the nucleotides guanosine penta- and tetraphosphate [(p)ppGpp] and adenosine penta- and tetraphosphate [(p)ppApp] have distinct functions in bacteria. (p)ppGpp mediates survival under nutrient-limiting conditions and its intracellular levels are regulated by synthetases and hydrolases belonging to the RelA-SpoT homolog (RSH) family of enzymes. By contrast, (p)ppApp is not known to be involved in nutrient stress responses and is synthesized by RSH-resembling toxins that inhibit the growth of bacterial cells. However, it remains unclear whether there exists a family of hydrolases that specifically act on (p)ppApp to reverse its toxic effects. Here, we present the structure and biochemical characterization of adenosine 3'-pyrophosphohydrolase 1 (Aph1), the founding member of a monofunctional (p)ppApp hydrolase family of enzymes. Our work reveals that Aph1 adopts a histidine-aspartate (HD)-domain fold characteristic of phosphohydrolase metalloenzymes and its activity mitigates the growth inhibitory effects of (p)ppApp-synthesizing toxins. Using an informatic approach, we identify over 2,000 putative (p)ppApp hydrolases that are widely distributed across bacterial phyla and found in diverse genomic contexts, and we demonstrate that 12 representative members hydrolyze ppApp. In addition, our in silico analyses reveal a unique molecular signature that is specific to (p)ppApp hydrolases, and we show that mutation of two residues within this signature broadens the specificity of Aph1 to promiscuously hydrolyze (p)ppGpp in vitro. Overall, our findings indicate that like (p)ppGpp hydrolases, (p)ppApp hydrolases are widespread in bacteria and may play important and underappreciated role(s) in bacterial physiology.

Micronuclei formation in rainbow trout cells exposed to multiple stressors: Morpholine, heat shock, and ionizing radiation.

Discharges from industrial cooling water systems can include low levels of morpholine (a chemical pH regulator and corrosion inhibitor), as well as transiently higher temperature effluent water which present a potential source of environmental impact to aquatic biota. The effects of environmental levels of morpholine or heat shock (HS) treatment alone and in combination with a challenge high-dose of 137Cs ionizing radiation were studied using the cytokinesis block micronucleus assay in a rainbow trout cell line (RTG-2). Morpholine treatment of 10 or 100mgL-1 alone produced no significant effects, and no interaction was observed in combination with 7.75Gy radiation. A 9°C magnitude HS treatment alone significantly increased micronuclei formation. A synergistic response was observed when 9°C HS was combined with 7.75Gy radiation, with 15% more cells containing 3 or more micronuclei than the sum of each individual stressor. A synergistic increase in the average number of micronuclei was observed when morpholine and a 9°C HS were co-treated. These results indicate that morpholine at environmentally-relevant levels does not impact micronuclei formation or cell cycle progression however 9°C HS may be of potential concern both alone and in combination with other stressor treatments.