Abstract
1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany
2 Institut für Mikrobiologie, Technische Universität München, Am Hochanger 4, D85354 Freising, Germany
3 Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany
Ralstonia eutropha H16 is probably the best-studied Knallgas bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB–4 and ΔphaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB–4 and (iv) cells from the transition phase of R. eutropha ΔphaC1 with those from the transition phase of R. eutropha PHB–4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB–4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB–4. In strain ΔphaC1, phaA and phaB1 were highly induced compared with PHB–4. Additionally, the results of this study suggest that mutant strain PHB–4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB–4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB–4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16ΔphaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.