Functional analysis of the 5′ regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-d-nicotine oxidase gene

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Abstract
A functional analysis of the Arthrobacter oxidans 6-hydroxy-d-nicotine oxidase (6-HDNO) gene promoter (−35 region TTGACA and −10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis. Deletion of the C residue from the −10 promoter region or mutations introduced upstream of the −10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E. coli and A. oxidans coupled transcription-translation systems in vitro. From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A. oxidans harbours an RNA polymerase functionally homologous to the E. coli σ70 and Bacillus subtilis σ43 polymerases. Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E. coli. This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus −10 region and to 1.7 in the case of the tac promoter. A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange. The failure of cAMP to stimulate 6-HDNO expression in the A. oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression. ppGpp, a nucleotide involved in the initiation of morphological and physiological differentiation in stationary cells, did not significantly affect 6-HDNO expression in the in vitro transcription-translation system.