The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida
Article first published online: 14 MAR 2014
© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd
Environmental Microbiology Reports
How to Cite
Jiménez-Fernández, A., López-Sánchez, A., Calero, P. and Govantes, F. (2014), The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida. Environmental Microbiology Reports. doi: 10.1111/1758-2229.12153
- Article first published online: 14 MAR 2014
- Accepted manuscript online: 15 FEB 2014 06:00AM EST
- Manuscript Accepted: 31 JAN 2014
- Manuscript Received: 30 JAN 2014
- Spanish Ministerio de Ciencia e Innovación. Grant Number: BIO2010–17853
Fig. S1. Complementation of the biofilm dispersal phenotype. Microtiter plate cultures of the wild-type KT2442 and the ΔbifA mutant bearing the empty miniTn7Ω-Gm transposon (Ø) or the miniTn7Ω-bifA transposon expressing bifA (+BifA) were grown in LB or K10T-1 medium and planktonic and biofilm growth was monitored for 24 or 48 h.
A and B. Planktonic (A) and biofilm (B) growth of cells on 96-well microtiter plates containing LB. Samples were measured at 8 h (open bars) and 24 h (closed bars).
C and D. Planktonic (C) and biofilm (D) growth of cells on 96-well microtiter plates containing K10T-1. Samples were measured at 8 h (open bars), 24 h (light shaded bars), 32 h (dark shaded bars) and 24 h (closed bars). Bars represent the averages and standard deviations of at least three independent experiments.
Fig. S2. Swimming and swarming assays of the wild-type and ΔbifA strains.
A. Swimming assays showing a picture of a typical swim plate with the wild-type and ΔbifA strains, and a non-motile fleQ mutant used as a control (right), and a plot showing the diameter of the swimming zone of the ΔbifA mutant relative to that of the wild type, set to 100% (left). The plot shows the average and standard deviation of at least three independent assays.
B. Swarming assays showing pictures of representative swarming plates of the wild-type (left) and ΔbifA strains (right).
Fig. S3. Alignment and conservation of the BifA GGDEF and EAL domains.
A. Alignment of the P. putida KT2442 and P. aeruginosa BifA GGDEF domains. Letters on top indicate the location of the signature GGDEF motif, and residues involved in interaction with GTP (red), dimerization (blue), catalysis (green) and allosteric inhibition (orange) (Chan et al., 2004).
B. Alignment of the P. putida KT2442 and P. aeruginosa BifA EAL domains. Letters on top indicate the location of the signature EAL motif, and residues conserved in over 80% of active c-di-GMP PDEs (Schmidt et al., 2005). Residues essential for PDE activity are shown in red (Rao et al., 2008). Alignment was performed using ClustalW2 at the EMBL-EBI server (Larkin et al., 2007; Goujon et al., 2010).
Fig. S4. Effect of mutations at the EAL and GGDQF motifs of BifA on biofilm dispersal. The wild-type KT2442 and the ΔbifA mutant bearing the empty miniTn7BB-Gm transposon (Ø) or its derivatives expressing FLAG-tagged versions of the wild-type BifA (+BifA), or its mutant derivatives (+BifA EAL-AAL; +BifA GGDQF-AAAQF; +BifA GGDQF-GEDQF). Top: biofilm growth on 96-well microtiter plates containing LB at 8 h (open bars) and 24 h (closed bars). Bars represent the average and standard deviation of at least three independent assays. Bottom: Western blots from cells of the strains used in the top panel using anti-FLAG monoclonal antibodies. The arrowheads indicate the tagged BifA bands. Non-specific bands are shown as loading controls.
A and B. Results of separate sets of assays performed with the EAL and GGDQF mutants respectively.
Table S1. Bacterial strains, plasmids and oligonucleotides used in this work. Underlined bases indicate oligonucleotide positions that differ from the corresponding templates.
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