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. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture 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 degreesC in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.