Primer extension analysis of tcbC gene transcription in the heterologous host E. coli. (A) Total RNA was isolated from E. coli RM221 (Δpfl Δact) containing different mutant derivatives of the pMU10 plasmid. A total of 15 μg of RNA was used for each primer extension experiment. The nucleotide sequence shown was determined with the same oligonucleotide primer with pMU10 as DNA template as that used in the primer extension reactions. Lane 1, RM221/pMU10 grown aerobically; lane 2, RM221/pMU10 grown anaerobically; lane 3, RM221/pMU1011 grown anaerobically; lane 4, RM221/pMU1012 grown anaerobically; lane 5, RM221/pMU1013 grown anaerobically. The arrow indicates the 5′ end of the tcbC mRNA transcript. (B) The location in the DNA sequence of the 5′ end of the tcbC transcript is indicated by the shorter arrow. Putative −10 and −35 RNA polymerase binding sites are underlined, and the first two amino acids of the TcbC protein are shown. The ochre termination codon of the preceding tcbB gene is boxed.