Figure 1
B. bronchiseptica SigE is a functional sigma factor. (A) Amino acid sequence alignment of RpoE- like sigma factors from Escherichia coli (Ecoli), Vibrio cholerae (Vchol), Pseudomonas aeruginosa (Paer), Nitrosomonas europaea (Neur) and B. bronchiseptica (Bbron) using ClustalW2 (EMBL-EBI). Asterisks indicate identity, two dots indicate strong similarity, and one dot indicates weak similarity between amino acid residues. Conserved sigma factor regions 2.1-2.4 and 4.1-4.2 [22] are indicated above the alignment. Regions 2.3, 2.4, and 4.2 are responsible for promoter recognition [22]. (B) β-galactosidase activity from the E. coli rpoHP3-lacZ reporter increases when B. bronchiseptica sigE expression is induced from plasmid pSEB006 in strain SEA5005 by the addition of IPTG. No increase is seen upon IPTG addition to the control strain, SEA008, containing the empty vector. The observed difference in the amount of β-galactosidase activity between the two strains in the presence of IPTG is statistically significant (P value <0.001) (C) In vitro transcription from a supercoiled plasmid template containing the E. coli σE-dependent rpoHP3 promoter with E. coli core RNA polymerase (core), SigE alone, EσE, and ESigE (left panel). In vitro transcription from a linear template containing the promoter region of B. bronchiseptica fam, with E. coli core RNAP alone (core), or ESigE (right panel). Arrows indicate transcripts from the rpoHP3 and fam promoters. Below, an alignment of the E. coli rpoHP3 and B. bronchiseptica fam promoter sequences and a sequence logo showing the consensus promoter for RpoE-like (ECF02) sigma factors from Staron et al. [24].