Construction of E. coli Reporter Strains for the Study of Stress-Answer Regulator Proteins

Escherichia coli K12 is a model organism in microbiology and genetics. It is currently also used for production of valuable compounds such as aminoacids, vitamins, or fine chemicals. During bioreactor cultivations, however, the cells often recognice varying conditions of aeration (aerobiosis, microaerophilic or anaerobic), of carbon source supply (glucose, glycerol, acetate), or nitrogen. To study the response of E. colicells to these varying conditions, we have constructed defined reporter strains.

First, knock-out strains for major regulator proteins of C-, N- and O- regulation (GlnK, GlnB, ArcA, Fnr, OxyR, Mlc, Crp, Crr, Cra, RpoS, RpoN) [1] have been provided by the method of Datsenko & Wanner [2] and were characterized. Next, we compared various reporter genes (lacZ encoding ß-galactosidase, gus for a ß-glucuronidase [3], gfp, or bfp for an oxygen-independent blue fluorescent protein from a metagenomic source [4]) for their applicability as C-; N-, O- monitors. We took care to have universal and stable reporter systems. Thus, apart from plasmid-borne reporter genes, we also are working on the chromosomal integration to avoid genetic instabilities and the use of antibiotic resistance markers. To assay the applicability of the reporter systems, we fused well-known promoter regions which are under the control of a given regulator (C-, N- or O-regulons) and integrated them as single copy into the chromosomal DNA of E.coliwild-type strain W3110 and of the respective regulator mutant strains.

Reporter strength was then measured by standard enzyme assays for the chromogenic substrates (ONPG for LacZ, PNPG for Gus) or for fluorescence (GFP, BFP) using a laser fluorescence microscopy and fluorophotometric measurements. Results will be presented and discussed.

[1] A. Martinez-Antonio (2011) Network Biology 1(1):21-33

[2] K.A. Datsenko, B.L. Wanner (2000) Proc.Natl.Acad.Sci. USA 97, 6640-45.

[3] C. Platteeuw al. (1994) Applied and Environmental Microbiology, 587-593

[4]  Chang-Sun Hwang et al. (2012) Biochemical and Biophysical Research Communications Vol 419, 676-681