Complementation in E. coli

pQE80 His-Hfq3 expression plasmids (Additional file 1: Table S2) were transformed into a ΔlacZΔhfq strain of E. coli carrying an rpoS-lacZ translational fusion (strain YN585). Resulting strains were streaked to MacConkey (+100 μg/mL ampicillin) agar plates (BD Difco) and incubated for ≈ 16 h at 37 °C to observe color development. Lactose in the MacConkey plates induces constitutive expression of His-Hfqs from the plasmids. β-galactosidase assays were conducted on overnight MacConkey plate scrapings essentially as described in reference [18]. To observe total Hfq protein expression levels, cells were scraped from MacConkey plates and lysed with an 8 M urea buffer (pH 8). Lysates were visualized on 16% Tricine gels by Coomassie Blue staining.

We observed that deletion of the His-tag from the wild-type Hfq3 construct reduced the pink color on MacConkey plates; this may be due to reduction in protein accumulation in the absence of the tag, impacts on other properties of heterologous expression, and/or impacts of the tag on the biochemistry of the protein itself. Therefore, the original His-Hfq3 system for heterologous production in E. coli was retained. (We note that all Bacillus phenotypes described in this paper are in the absence of any additional tag residues, with the native protein sequence).