Figure 6
Localization of fluorescent proteins in P. putida and E. coli strains. Cells shown are representative of >6 fields viewed in two or three independent experiments. Upper panels (A to D) and cognate western analysis are of strains cultured on LB. (A) P. putida KT2701 (negative control; cells examined n = 735). (B) P. putida KT2701::aer2-eyfp (positive control, mono-copy chromosomal fusion; cells examined n = 342 of which 64% exhibited polar localization). (C) P. putida KT2701::pp4397-eyfp (mono-copy chromosomal fusion; cells examined n = 219). (D) P. putida KT2701::pp4397-eyfp/Δpp2258 (PP2258 null with elevated c-di-GMP; cells examined n = 649). Western analysis of EYFP-tagged proteins expressed from mono-copy chromosomal translational fusions present in 50 and 25 µg of crude extract. Cell were harvested for Western analysis at the same time as fixing for imaging (after 2 to 2.5 hrs of growth; OD600 0.5 to 0.7), which contrasts those shown for the motility assays in Fig. 5 (harvested after 5 hr of growth; OD600 ~3.5). Note that Aer2-EYFP, although clearly visible at the pole of the cell in panel B, is expressed at much lower levels than PP4397-EYFP and is not detected at the exposure shown. The cropped image is derived from the same experiment processed in parallel on the same gel, and are shown alongside molecular size markers in Fig. S6A. Lower panels [E to H] and cognate western are of strains cultured on LB in the presence of 1% L-arabinose. Boxed images are differentially exposed cells for comparison of the presence or lack of puncta. (E) P. putida KT2701-Δpp2258/Δpp4397 (double PP2258/PP4397 null strain) carrying the multi-copy araC/PBAD pp4397-eyfp expression plasmid (pVI2374). Cells examined: n = 271, 0% with puncta. (F) P. putida KT2701-Δpp2258/ΔPP4397 carrying the multi-copy araC/PBAD ycgR-eyfp expression plasmid (pVI2375). Cells examined n = 282, 0% with puncta. (G) E. coli MG1655-ΔyhjH/ΔycgR (double YhjH/YcgR null strain) carrying the multi-copy araC/PBAD pp4397-eyfp expression plasmid (pVI2374). Cells examined: n = 343, 0% with puncta. (H) E. coli MG1655-ΔyhjH/ΔycgR carrying the multi-copy araC/PBAD ycgR-eyfp expression plasmid (pVI2375). Cells examined: n = 252, 25% with puncta [1 to 3 per cell]. Western analysis of EYFP-tagged proteins expressed from multi-copy translational fusions present in 25 and 12.5 µg of crude extract from P. putida (left) and E. coli (right). Cropped images are derived from the same experiment processed in parallel on the same gel, and are shown alongside molecular size markers in Fig. S6B.
