Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments

Chloroform-soluble material was extracted from two strains of L. pneumophil a serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonan ce spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB w as located in electron-dense intracellular inclusions, which fluoresced bri ght yellow when stained with the lipophilic dye Nile red. A Nile red spectr ofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited c ulture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24 degrees C in a low-nutrient tap water environment. Nil e red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PWB utilization, as revealed by the declin e in mean cellular fluorescence and cell complexity, correlated with loss o f culturability. Fluorescence microscopy provided visual evidence of PHB ut ilization, with a marked reduction in the number of Nile red-stained granul es during starvation. Heat shock treatment failed to resuscitate noncultura ble cells. This study demonstrates that L. pneumophila accumulates signific ant intracellular reserves of PHB, which promote its long-term survival und er conditions of starvation.