†Present address: Instituto Universitario de Electroquímica. Universidad de Alicante. 03080- Alicante, Spain.
The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442
Article first published online: 14 APR 2010
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Pseudomonas. Editors: Professors Burkhard Tummler, Victor de Lorenzo, Alain Filloux and Joyce Loper
Volume 12, Issue 6, pages 1591–1603, June 2010
How to Cite
De Eugenio, L. I., Galán, B., Escapa, I. F., Maestro, B., Sanz, J. M., García, J. L. and Prieto, M. A. (2010), The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442. Environmental Microbiology, 12: 1591–1603. doi: 10.1111/j.1462-2920.2010.02199.x
- Issue published online: 3 JUN 2010
- Article first published online: 14 APR 2010
- Received 3 November, 2009; accept 22 January, 2010.
Fig. S1. Quantification of the expression rates of the pha genes by real-time RT-PCR assays. Gene transcription profiles of pha genes throughout growth of P. putida KT2442 (A–F), KT42C1 (G–L) and KT42D (M–Q) in M63 0.1 N plus 15 mM sodium octanoate (black bars) or 20 mM glucose (red bars) are shown; samples along the growth curve were taken at 0, 3.5, 7 and 23 h. The gene transcription of phaC1 (A, G, M), phaZ (B, H, N), phaC2 (C, I, O), phaD (D, J), phaF (E, K, P) and phaI (F, L, Q) genes is depicted. An initial concentration of 5 ng cDNA was used for quantitative RT-PCR. Error bars represent standard deviation calculated from the results of three independent experiments. y-axis: transcription level (cDNA ng); x-axis: time of culture (h).
Fig. S2. Gel retardation analyses of PhaD binding to the PI, PC1 and PF promoter regions.
A. The DNA probes used (PI, PC1 and PF) are indicated schematically on the right. Cell extract and gel retardation analyses were performed as described under Experimental procedures. Lanes 2–7 contained 0.086, 0.17, 0.34, 0.69, 1.7 and 3.4 μg of total protein of PhaD+ extracts obtained from cells bearing plasmid pIZD (phaD) respectively. Lane 1 contained no extract and lane 8 contains 3.4 μg of total protein of PhaD-free extracts obtained from cells bearing control plasmid pIZ1016 respectively. The DNA–PhaD complexes are indicated.
B. PhaD binding competition analysis to PI promoter with unlabelled DNA-specific probe (lanes 3, 4, 5) and unrelated salmon sperm DNA (lanes 6, 7, 8). Gel retardation was performed in the presence of 1.7 μg of total protein of PhaD+ extracts. Lane 1 contains no extract.
Fig. S3. Homology-based 3D model of dimeric PhaD. A view of the most representative predicted polar interactions between HOCoA and PhaD. Colour scheme is CPK, with the exception of carbon atoms from the protein (green) or from the ligand (magenta). Oval highlights the 3-hydroxyoctanoyl moiety.
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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.