Figure 10
Regulation of orthologous target genes in native backgrounds. A-C: Sequences upstream of adhE, gltB and lrp orthologs. In each case, the sequence ends with the initiation codon. Lrp-binding sites and the transcriptional +1 position are known for E. coli K-12 [112]. Demonstrated Lrp binding sites are in underlined lowercase italics, and the -35 and -10 sequences inferred from the known +1 position (for E. coli) are boxed. Putative binding sites, predicted by the PRODORIC virtual footprinter [102] are shaded, and the match scores for predicted sites are shown to the right. For E. coli PgltB, one of the predicted sites overlaps an actual site, and gives a particularly high match score, though an overlapping actual site in Plrp does not. V. cholerae has two nearly-tandem copies of the gltBD operon on chromosome I. The 5'-most gltB isozyme ("Vch1", locus tag Vc2373) is 43% identical to Eco gltB, while the 3'-most isozyme ("Vch2", Vc2376) is 73% identical to Eco gltB in amino acid sequence. D-E: Samples were isolated at an OD600 nm of 0.3 (log), as well as 1 h after linear growth stopped (stationary), from E. coli, P. mirabilis and V. cholerae wild-type and lrp cultures growing in MOPS defined rich medium. QRT-PCR was used to determine the relative levels of adhE, gltB and recA messages, with recA serving to provide a Lrp-independent baseline. The experiment was performed in triplicate and the level of message was determined using the standard curve method and normalization to recA. D – adhE. E – gltB. For each plot filled symbols represent log phase levels and open symbols represent stationary phase levels. The symbol shapes indicate the species: P. mirabilis (triangles) E. coli (circles) or V. cholerae (Vc2373, squares). The line indicates the position for data if no effect of Lrp is seen (ratio of 1); points above the line are consistent with repression, while those below the line are consistent with activation by Lrp. The dotted lines show, to facilitate comparison, the borders of a twofold effect.