Application of pheB as a reporter gene for Geobacillus spp., enabling qualitative colony screening and quantitative analysis of promoter strength

Abstract

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism.

Fig 1

(A) E. coli-Geobacillus sp. shuttle vector pUCG18 with the multiple-cloning site (MCS) containing the pheB gene from G. stearothermophilus DSMZ 6285 cloned downstream of the ldh promoter from G. stearothermophilus NCA 1503. (B) pldh activity assessed by C23O in DL44 (Δldh)-pGR002 cell extracts. Aerobic, 50 ml of media in a cotton wool-sealed 500-ml Erlenmeyer flask. Anaerobic, 12 ml of media, sparged for 5 min with filter-sterilized N₂, in a 15-ml Hungate anaerobic growth tube. Microaerobic, 50 ml of medium in a gas-impermeable 250-ml Erlenmeyer flask with a Suba-Seal cap. (C) Levels of pheB transcript measured by qRT-PCR in aerobic and microaerobic cultures and under anaerobic conditions. The kanamycin resistance gene acts as an endogenous control. The lowest transcript levels were arbitrarily set to 1 and used as a reference for fold change calculation. (D) C23O activity determined in samples from chemostat cultures grown on 55 mM glucose at 55°C and pH 7.