FIG 3.
Enzyme activity assays with M. thermautotrophicus ΔH strains that carry a thermostable β-galactosidase (BgaB)-encoding gene under the control of four distinct promoter sequences. (A) Sequence alignment of distinct putative promoter sequences that we analyzed for activity to drive the expression of a thermostable β-galactosidase (bgaB) gene. Sequence repeats in Pmrt(M.t.) are underlined. The transcription start site is indicated by “+1,” highlighted in bold, and underlined. TATA box sequences of Psynth, Psynth(BRE), and PhmtB are surrounded by a box. BRE sequences are highlighted in italics and ribosome-binding sites in red. Dashes are used as spacers, while dots indicate additional base pairs, which are left out here for visualization. Differences between Psynth, Psynth(BRE), and PhmtB between the TATA box sequence and transcription start site are highlighted in bold. (B) Qualitative analysis of BgaB activity with S-Gal as chromogenic substance in an in vitro assay with cell lysate of empty-vector-carrying M. thermautotrophicus ΔH (pMVS-V1) or pMVS1111A:Psynth-bgaB-carrying M. thermautotrophicus ΔH (Psynth-bgaB) cells. (C) Quantitative analysis of BgaB activity with ONPG as chromogenic substance in an in vitro assay with cell lysate of M. thermautotrophicus ΔH strains that carry plasmids with the bgaB gene under the control of the four distinct promoters [pMVS-V1, empty-vector control; Pmrt(M.t.)-bgaB, pMVS1111A:Pmrt(M.t.)-bgaB; Psynth-bgaB, pMVS1111A:Psynth-bgaB; Psynth(BRE)-bgaB, pMVS1111A:Psynth(BRE)-bgaB; PhmtB-bgaB pMVS1111A:PhmtB-bgaB]. Average (n = 3) with error bars indicating standard deviation. Significance was tested with Student’s t test (two-tailed): *, significant difference (P < 0.05); **, highly significant difference (P < 0.01); n.s., no significant difference (P > 0.05).