An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance - PubMed (original) (raw)

An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance

Brittany A Morrison et al. Biol Lett. 2008.

Abstract

Disparate psychrophiles (e.g. glacier ice worms, bacteria, algae and fungi) elevate steady-state intracellular ATP levels as temperatures decline, which has been interpreted as a compensatory mechanism to offset reductions in molecular motion and Gibb's free energy of ATP hydrolysis. In this study, we sought to manipulate steady-state ATP levels in the mesophilic bacterium, Escherichia coli, to investigate the relationship between cold temperature survivability and elevated intracellular ATP. Based on known energetic pathways and feedback loops, we targeted the AMP nucleotidase (amn) gene, which is thought to encode the primary AMP degradative enzyme in prokaryotes. By knocking out amn in wild-type E. coli DY330 cells using recombineering methodology, we generated a mutant (AMNk) that elevated intracellular ATP levels by more than 30% across its viable temperature range. As temperature was lowered, the relative ATP disparity between AMNk and DY330 cells increased to approximately 66% at 10 degrees C, and was approximately 100% after storage at 0 degrees C for 5-7 days. AMNk cells stored at 0 degrees C for 7 days displayed approximately fivefold higher cell viability than wild-type DY330 cells treated in the same manner.

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Figures

Figure 1

Figure 1

AMNk knockout cells elevated steady-state ATP levels compared with wild-type E. coli cells. ATP levels decreased in all mesophilic cells as temperature deviated from 37°C (line graph), but the relative difference in ATP levels between AMNk and DY330 increased (bars). The psychrophilic bacterium, _V_sp1, increased [ATP] as temperature decreased.

Figure 2

Figure 2

Survivability of AMNk versus DY330 cells after cold storage at 0°C. AMNk cells displayed an approximately 2.5- and approximately 5-fold increase (p<0.05) in cold survivability compared with DY330 after 5 and 7 days cold storage, respectively. Data are normalized and presented as a fraction of cells at zero time. ATP levels fell steadily in both AMNk and DY330 cells as a function of time, but were approximately 100% higher in AMNk after 5 days (0.99±0.02 versus 0.48±0.02 pmol ATP mg−1) and 7 days (0.85±0.01 versus 0.41±0.02 pmol ATP mg−1; dashed line, right axis; cf. figure 1).

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