Fine structural analysis of the Zoogloea ramigera phbA-phbB locus encoding beta-ketothiolase and acetoacetyl-CoA...

A series of expression plasmids containing either the complete insert from plasmid pUCDBK1 (Peoples et al., 1987) or sub-fragments thereof were constructed in a tac promoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH-specific acetoacetyl-CoA reductase in the 2.3kb of sequence located downstream from the beta-ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1kb) of this region was determined. An open reading frame was located 88bp downstream from the stop codon of the thiolase gene encoding a potential polypeptide of Mr 25,000, which is in good agreement with that observed for the overexpressed protein on SDS-PAGE. N-terminal protein sequence data obtained by Edman degradation of the purified Mr = 25,000 polypeptide were used to identify the correct start of the NADPH-specific acetoacetyl-CoA reductase gene. Hence in Z. ramigera, the genes encoding beta-ketothiolase (phbA) and NADPH-specific acetoacetyl-CoA reductase (phbB) are organized as phbA-phbB. S1-nuclease analysis of Z. ramigera RNA identified a transcription start site 85 bp upstream from the phbA structural gene locating the promoter region.