Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

Abstract

bPoly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor 54 increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

“…The PHB synthase PhaC1 was only the sixth-most-abundant protein. Similar differences in the expression of PHB synthase and phasins were determined by transcriptional analysis or quantitative Western blot analysis in previous works (41,(58)(59)(60). However, the culture conditions and the time points of sampling were different from those in our study.…”

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

“…The structural genes for the formation of PHA, as well as the genes responsible for the regulation and mobilization of PHA under C-limiting conditions, have been identified and biochemically characterized over the past few decades (for a selection of reviews, see references 3 and 15-20). Much evidence that the key enzymes of PHA synthesis (PHA synthase [PhaC]) and PHA mobilization (PHB depolymerases [PhaZs]) are constitutively expressed and are generally present on the carbonosome granules has been published (21)(22)(23)(24). This raises the question of how the synthesis and mobilization of carbonosomes are regulated and how a futile cycle of simultaneous synthesis and degradation is avoided.…”

EliA is required for inducing the stearyl alcohol-mediated expression of secretory proteins and production of polyester in Ralstonia sp. NT80

“…Transcription of the operon responsible for the PHA biosynthesis in NT80 cells was, however, not significantly upregulated by stearyl alcohol (results not shown). In this regard, it is noteworthy that the transcription level of the phaCAB gene cluster of R. eutropha did not change significantly under the conditions where PHA is produced or utilized (Brigham et al, 2012). Under nitrogen-deficient conditions, the biosynthesis of PHA in Paracoccus denitrificans is also not regulated at the mRNA or protein level, but depends on the amount of acetyl-CoA (Kojima et al, 2004).…”