The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440
Article first published online: 18 AUG 2013
© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology
Environmental Microbiology Reports
Volume 5, Issue 5, pages 740–746, October 2013
How to Cite
Casey, W. T., Nikodinovic-Runic, J., Fonseca Garcia, P., Guzik, M. W., McGrath, J. W., Quinn, J. P., Cagney, G., Prieto, M. A. and O'Connor, K. E. (2013), The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440. Environmental Microbiology Reports, 5: 740–746. doi: 10.1111/1758-2229.12076
- Issue published online: 2 OCT 2013
- Article first published online: 18 AUG 2013
- Accepted manuscript online: 19 JUN 2013 06:39AM EST
- Manuscript Accepted: 11 JUN 2013
- Manuscript Received: 26 APR 2013
- IRCSET (IRC) EMBARK
- University College Dublin under the Lord Edward Fitzgerald Memorial Fund
- Ministerio de Economia y Competitividad. Grant Number: BIO2010-21049
Appendix S1. Experimental Procedures.
Fig. S1. Growth and PHA accumulation for P. putida KT2440 wild type (A) and Δppk (B) over 48 h grown in MSM-limited nitrogen medium supplemented with 20 mM of glucose.
Fig. S2. CDW (A) and PHA (B) accumulation in P. putida KT2440 wild type, Δppk and Δppk pJb861/ppk incubated in MSM-limited nitrogen medium supplemented with either 20 mM of glycerol with 2 mM m-toluic of acid for induction of pJB861/ppk expression vector over 48 h. All values are relative to wild-type levels and are displayed as percentages of wild-type CDW and PHA.
Fig. S3. Biofilm formation by P. putida KT2440 wild type and Δppk over 24 and 48 h grown in static 96 well plates with MSM media supplemented with 0.4% glucose.
Fig. S4. Comparison of motility (colony diameter) of P. putida KT2440 wild type and Δppk grown on semi-solid (0.25%) Luria–Bertani (LB) agar plates over 24 h at 20°C, 30°C and 37°C.
Fig. S5. Southern blot image of P. putida KT2440 wild-type and Δppk genomic DNA. Lane 1 contains digoxigenin-labelled DNA molecular weight marker. Lanes 2 and 3 contain mutant genomic DNA digested to completion with an NcoI/NdeI double restriction digest and NcoI single digest respectively. Lane 4 (no band visible) contains wild-type genomic DNA digested to completion with NcoI. Blot was hybridized with a digoxigenin-labelled gentamicin probe.
Fig. S6. DNA sequence of the flanking regions around the incorporated gentamicin cassette in genomic DNA extracted from KT2440 Δppk. It should be noted that the sequence is given for the genomic minus strand. Sequence is a result of the overlapping of nucleotide reads from the pimers ppk_seq_(F) and ppk_seq_(R) (Table S2) carried out by SourceBioscience (Ireland). Relative positions of these primers are marked in blue; however, accurate sequencing began at the locations marked with arrows. The gentamicin cassette is highlighted as CAPITAL letters. HindIII restriction site due to SDM are underlined. Small fragments (31 and 36 bases) of the ppk gene remain and are highlighted in red. Sequence also shows flanking regions on both the 5′ (762 bp) and the 3′ (760 bp) ends of the ppk gene.
Table S1. Protein abundance in P. putida KT2440 wild type and Δppk was estimated by frequency of tandem mass spectrometry events (Spectral Count) for each protein associated with polyphosphate and glycerol at early (OD540 0.2), mid (OD540 0.6) and late log (OD540 1) stage of growth. A selection of ribosomal proteins is displayed to highlight that expression of these proteins is not affected by the ppk deletion.
Table S2. List of primers used in this study.
Table S3. Bacterial strains used in this study.
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