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Fig. 5 | Microbial Cell Factories

Fig. 5

From: Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

Fig. 5

a Analysis of transcription products of kstD genes in R. ruber strain Chol-4 by specific RT-PCR amplification and agarose gel electrophoresis. RNA samples were isolated from cultures grown in: M457 minimal medium supplemented with sodium acetate (lane NaAc), AD (lane AD) or cholesterol (lane CHO) and LB medium supplemented with (lane LB + AD) or without AD (lane LB). RT—refers to negative controls (not incubated with retrotranscriptase) to exclude DNA contamination in RNA samples. Primers used in each PCR reaction and amplified fragment size are shown in brackets. As non-induced expression controls, the transcription of two R. ruber Chol-4 enzyme genes was followed in the same conditions: ORF4, that codes for a FAD-binding dehydrogenase D092_14375 (99% identity with the protein NCIB::WP_010594120.1 of Rhodococcus sp. P14) and ORF5 (D092_13875) that codes for a fumarate reductase (99% identity with NCIB::WP_010594021.1 of Rhodococcus sp. P14; see sequence in Additional file 6). the FAD-binding dehydrogenase D092_14375 and the fumarate reductase D092_13875. b RT-PCR assays of R. ruber Chol-4 kstD1 and kstD3 transcription carried out in kstD2 R. ruber mutant growing in: LB (lane LB) or M457 plus CHO (lane CHO), NaAc and AD (lane NaAc + AD) or NaAc (lane NaAc). Lane DNA shows the result of the control experiment: PCR made on R. ruber DNA

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