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Fig. S1. Schematic representation of the GGDEF, EAL and HD-GYP domain proteins in P. putida KT2440. Data extracted from http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html (Galperin et al., 2010).

Fig. S2. Transcription from the aer1-PP2258 promoter is unaffected by elevated c-di-GMP.

A. Schematic representation of the region upstream of the aer1-PP2258 operon with the intergenic region, but not the genes, drawn to scale. The DNA sequence of the operon promoter region is given at the top with the −35 (TTTATT, consensus TTGACA) and −10 (TAGAAT, consensus TATAAT) motifs highlighted in bold. The shuffled −10 sequence (aAagtT) present in pVI1057 and pVI1058 is indicated by an asterisk (*). The DNA regions 1–5 show the extent of the intergenic DNA present in in vitro transcription templates and luciferase transcriptional reporter plasmids used in B and C.

B. In vitro multiple round transcription assay with 25 nM σ70-RNAP and supercoiled plasmid templates (10 nM) carrying different regions upstream of aer1-PP2258 (see A). Assays were performed as described in Bernardo and colleagues (2006) using T-buffer (35 mM Tris-Ac pH 7.9, 70 mM KAc, 5 mM MgAc2, 20 mM NH4Ac, 1 mM DTT and 0.275 mg ml−1 BSA). Transcripts produced from the aer1-PP2258 promoter are indicated by black arrows – upper arrow for region 1, lower arrow for regions 2–5. The upper grey arrow indicates the transcript from a control plasmid, pVI948, that carries the σ70-dependent Pr promoter with 8 nucleotides (+8) downstream of the transcriptional start, while the lower grey arrow indicates the transcripts produced from the vector located RNA1 promoter, which is present on all templates and serves as a loading control.

C. In vivo luciferase plate assays with P. putida strains harbouring transcriptional reporter plasmids with the promoter-less luxAB genes under control of the different aer1-PP2258 upstream regions (see A). Cells were grown overnight at 30OC on LB plates containing carbenicillin and the results recorded on X-ray film after addition of decanal (100 μl of 1:200 dilution) to the lid of inverted plates. The left-hand image shows luciferase activity of KT2701 harbouring the indicated reporter plasmid or the cognate pVI928 vector control (vec.). The right hand image shows luciferase activity of the same strains that also harbour either the lacIQ/Ptac-PAS-GGDEF I-site mutant expression plasmid pVI1059 (+) or its cognate vector control pVI898 (−). Expression from the lacIQ/Ptac promoter of pVI1059 (and hence elevated intracellular levels of c-di-GMP) was induced by including 0.5 mM IPTG in the medium.

Fig. S3. Transcription from the aer1-PP2258 regulatory region is unresponsive to c-di-GMP.

A. Schematic representation of the region upstream of the aer1-PP2258 operon within luciferase transcriptional reporters, as in Fig. S2.

B–D. The graphs show growth (open symbols) and luciferase activity profiles (closed symbols) of LB-cultured P. putida KT2701 (native c-di-GMP levels; B), its PP2258 null counterpart (C), or KT2701 expressing the PAS-GGDEF I-site mutant from plasmid pVI1059 (highly elevated c-di-GMP levels; D). Resident reporter plasmids present in the three strains are as indicated by the symbol code in (A). Cells were grown at 30°C to exponential phase prior to a second dilution and initiation of the experiment. Light emission from 100 μl of whole cells, after addition of 1:2000 dilution of decanal, was measured using an Infinite M200 (Tecan) luminomiter. Specific activity is expressed as relative luciferase units per OD600 of 1.0. Data are the average of at least two independent cultures for each strain ± standard errors.

Table S1. Plasmids used in this study.

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