Figure 2
Transcription of PP4397 is dependent on both σ54 and σFliA. (A) Comparison of the genomic context of E. coli ycgR and P. putida flgZ/pp4397 genes. Upper schematic, illustration of E. coli MG1655 flgA, flgMN and ycgR genes (shown in black) with their cognate promoters indicated. Divergently transcribed genes are shown in grey. Lower, similar schematic of the P. putida flgA, flgM, flgN-like pp4396 and flgZ/pp4397 genes shown in black and the divergently transcribed cheV-3 and pp4398 genes shown in grey. The locations of primer pairs used for analysis of co-transcription of genes as depicted in panel B are indicated. (B) Agarose gels of PCR reactions using the indicated primer pairs (panel A) on P. putida genomic DNA, cDNA, or control samples where no reverse transcriptase was added to the cDNA reaction mixture (-RT). (C) In vivo transcription from PflgM in wild type P. putida KT2440 (1) and its FliA null (2) and RpoN null (3) counterparts carrying mono-copy transcriptional fusions to the promoter-less luxAB reporter genes (PP3733 to PP3735, Table S1). Graphed values are the average +/− standard deviation of six independent determinations from cultures grown to the stationary phase in LB (OD600 ~5.0 for wild-type and FliA null; ~2.1 for RpoN null). (D) Single-round in vitro transcription assays using 10 nM supercoiled DNA templates harbouring the σFliA –dependent P. putida Paer2 promoter (4; pVI1011) or PflgM (5; pVI2368) in the presence of 10 nM σFliA-RNAP. Inset shows images from one of two independent experiments used to obtain the graphed average values (PflgM upper; Paer2 lower). A comparison of the Paer2 and PflgM promoter sequences with the optimal consensus27 for P. putida σFliA is shown to the right.
