Poly-3-Hydroxybutyrate in Legionella pneumophila , an Energy Source for Survival in Low-Nutrient Environments

Abstract

Chloroform-soluble material was extracted from two strains ofL. 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°C 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.

Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non‐culturable cells

“…In dd water, L. pneumophila was unable to metabolize its inclusions of poly-β-hydroxybutyrate (PHB) and lost its culturability much faster than in tap water, suggesting that during starvation, culturability is extended at the expense of PHB. In fact, depletion of PHB and loss of culturability correlate in starved L. pneumophila (James et al, 1999). In general, the presence of PHB in starved bacteria extends metabolic processes, correlates with reduced degradation of RNA and proteins, and provides a direct carbon and energy source, i.e.…”

“…MIFs show prominent cytoplasmic inclusions and are motile and highly infectious to cultured cells . L. pneumophila inclusions consist of poly-β-hydoxybutyrate, as determined by nuclear magnetic resonance, gas chromatography and mass spectrometry (James et al, 1999). The inclusions become fluorescent after staining with Nile red and are consumed during starvation (as determined by fluorometry and fluorescence microscopy), suggesting that they constitute a nutritional reserve (James et al, 1999).…”

Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37

“…Under inanition, bacteria may remain viable for a long time if appropriate intracellular endogenous reserves are available (Kolter et al 1993;Ruiz et al 2001). Poly-b-hydroxyalkanoate (PHA) is an intracellular storage form of carbon and energy in bacterial cells (Lee 1996) and provides carbon and energy during starvation (James et al 1999).…”

Identification and characterization of genes, encoding the 3‐hydroxybutyrate dehydrogenase and a putative lipase, in an avirulent spontaneous Legionella pneumophila serogroup 6 mutant

“…As previously reported, Legionella accumulates PHB granules as an endogenous reserve of energy (29). For bacteria other than Legionella, it has also been demonstrated that BdhA is one of the enzymes of the metabolism of PHB, involved in the first step of the degradation process (28).…”