Adenylate kinase of Salmonella typhimurium is involved in the maintenance of the energy charge during the uptake of the osmoprotectant glycinebetaine
Abstract
To cope with osmotic stress, many organisms accumulate proline (LP) and glycinebetaine (GB) to high concentrations, thereby equalizing their intracellular osmolarity with that of the environment. In Salmonella typhimurium, exogenously added LP, and notably GB, stimulate growth under otherwise inhibitory conditions. The uptake of LP and GB is mediated by the permeases ProP, an ion-linked symporter driven by the proton motive force, and ProU, belonging to the family of ATP-binding cassette transporters powered by ATP hydrolysis. At the expense of cellular energy, these porters attain at least 1000-fold concentration gradients. To study the origins of GB osmoprotection, I characterized a set of four mutants which were impaired in stimulation of growth by GB but not LP. A genetic, molecular and biochemical characterization showed that the mutations were alleles of adk, encoding adenylate kinase. This kinase catalyzes the reaction: ATP + AMP $\Leftrightarrow$ 2ADP. Because its K$\sb{\rm eq}$ is close to unity, the enzyme carries out the de novo synthesis of ADP from AMP and the buffering of ATP when ADP is abundant, which maintains the energy charge. Three out of the four isolated mutations may lower the translational efficiency of the adk message. Mutants exhibited a decreased in vitro adenylate kinase activity. To elucidate the role of adenylate kinase in GB osmoprotection, I examined the physiology of the adk mutants in relation to uptake and accumulation of GB. Results revealed that while transport and accumulation of GB were unaffected, the addition of GB caused growth stasis, and a decline of ATP and of the energy charge, possibly due to uptake which in an adk mutant depletes ATP. I found support for this model by showing that PEP, (which can be taken up by S. typhimurium) overcame the sensitivity to GB and also restored the ATP near the normal level. This conclusion was further supported by the observation that in a strain unable to catabolize maltotriose (malQ) the uptake of this substrate caused sensitivity to maltotriose which was exacerbated by an additional adk mutation. Results suggest that adenylate kinase is important for the maintenance of the energy charge during active transport.
Degree
Ph.D.
Advisors
Csonka, Purdue University.
Subject Area
Anatomy & physiology|Animals|Genetics|Microbiology
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