Effects of hemB Antisense RNA on δ-Aminolevulinic Acid Production in Rhodobacter sphaeroides

Abstract

Genes for β and α polypeptides of the light harvesting II complexes in Rhodobacter sphaeroides 2.4.1 are encoded by the pucBA operon. Their expression is highly regulated by oxygen level and light intensity. δ-Aminolevulinic acid (ALA) in this bacterium is synthesized by ALA synthase isozymes encoded by hemA and hemT. ALA is subsequently converted into porphobilinogen by the enzyme δ-aminolevulinic acid dehydratase (ALAD) encoded by hemB gene. Recombinant plasmid pAS704 carries the pucBA promoter (pucB’) to transcribe 5’ fragment of hemB gene in the opposite direction (resulting in the partial 5’-end of hemB antisense RNA). pAS704 was constructed based on the broad-host range plasmid pRK415, and introduced into R. sphaeroides 2.4.1 through conjugation. 2.4.1 (pAS704) exhibited ALA synthase activity at least five times higher than that of 2.4.1 (pRK415). However, the extracellular ALA concentration remained the same in both strains suggesting that ALA synthase might be inhibited by ALA post- translationally.