Mobilization of Poly(3-Hydroxybutyrate) inRalstonia eutropha

Abstract

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.The mechanism of degradation of denatured, exogenous, crystalline poly(3-hydroxybutyrate) (PHB) by extracellular PHB depolymerases has been extensively studied during the last decade (for a recent review, see reference 6). However, intracellular PHB (i-PHB) degradation, i. e., the mobilization of previously accumulated amorphous PHB, is poorly understood. The beneficial effect of accumulated PHB on survival in the absence of a carbon source has been described for several species, including Ralstonia eutropha (4), Legionella pneumophila (5), and Hydrogenophaga pseudoflava (15). Recently, a DNA sequence of a putative i-PHB depolymerase of R. eutropha H16 was determined (GenBank accession no. AB017612). However, the physiological relevance of this gene during mobilization of PHB has not been investigated.PHB-rich cells of R. eutropha H16 (DSM428) were resuspended and incubated in carbon-free mineral-salt medium (12) with (mobilization) or without (control) NH 4 Cl for 6 days. The number of viable cells, in CFU per milliliter, increased about fourfold in the presence of NH 4 Cl within 30 h and remained constantly high for 6 days ( Fig. 1). In contrast, the CFU/ml remained almost unchanged in the absence of a nitrogen source. The PHB content of strain H16 (assayed through gas chromatography [1]) rapidly decreased from 70 to Ϸ30% during the first day of mobilization and decreased further, below 20%, after 6 days (Fig. 1). The PHB content of the control decreased only very little. The CFU/ml of the PHB-free mutant PHB Ϫ 4 (DSM541) decreased rapidly by several orders of magnitude after 3 days regardless of the absence or presence of a nitrogen source. Similar results were obtained with other bacteria such as Acidovorax delafieldii (DSM50403), Alcaligenes faecalis (14), a Comamonas sp. (DSM6781) and two Paracoccus denitrificans strains (DSM1404 and DSM413); however, the amount of PHB accumulation and the increase of the CFU/ml during mobilization of PHB were not as pronounced for these bacteria as for R. eutropha (data not shown). We conclude that R. eutropha H16 and other bacteria are able to mobilize previously accumulated PHB and to use the degradation products for one or two cell divisions even in the absence of an exogenous carbon source. Very little PHB is mobilized if ...

Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

“…In vivo, the levels of PHB in wt and ⌬phaZ1 R. eutropha strains were monitored. PHB depolymerase activity in crude extract was also measured (4,14). The results from these studies suggested that at least one additional intracellular depolymerase was required to account for PHB utilization.…”

“…Previous studies demonstrated the presence of the intracellular depolymerase PhaZ1 in R. eutropha (4,14,15). In vivo, the levels of PHB in wt and ⌬phaZ1 R. eutropha strains were monitored.…”

“…The function of PhaZ1 in vivo has recently been addressed by two groups independently (4,14). Each group generated a ⌬phaZ1 in R. eutropha H16 and then examined the fate of accumulated PHB under different growth conditions.…”

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

“…The emulsion was heated to remove chloroform and dialyzed for 24 h against 0.01% (wt/vol) sodium deoxycholate at room temperature. Native PHB was isolated from W. eutropha H16 and purified by glycerol density gradient (10). The 3HB oligomers were prepared as described previously (42).…”

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

“…Only recently, three intracellular PHA depolymerases (PhaZ1, PhaZ2 and PhaZ3) and a 3HB-oligomer hydrolase (previously also designated PhaZ2), which hydrolyse PHAs and the cleavage products produced by PhaZs, were cloned and characterized in R. eutropha (Saegusa et al, 2001(Saegusa et al, , 2002Kobayashi et al, 2003;York et al, 2003). However, in contrast to the extracellular degradation of PHAs (Jendrossek et al, 1996Jendrossek & Handrick, 2002), relatively little is known about intracellular mobilization and degradation in PHA-accumulating cells (Handrick et al, 2000;Saito & Kobayashi, 2002).…”