Gene organization and transcription analysis of the Agrobacterium tumefaciens glycogen (glg) operon: Two transcripts for the single phosphoglucomutase gene

The gene organization and transcription of the Agrobacterium gig operon dif fer from those in other bacteria. Agrobacterium tumefaciens A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism. The nucleotid e sequence and gene organization of a region containing ADP-glucose pyropho sphorylase (glgC), glycogen synthetase (glgA), and phosphoglucomutase (pgm) genes have been previously described (A. Uttaro and R. A. Ugalde, Gene 150 :117-122, 1994). In this work we report that the glycogen phosphorylase (gl gP) and branching enzyme (g/gB) genes are located immediately upstream of t his region. The complete nucleotide sequences of the glgP and glgB genes we re obtained, and mutants were constructed by targeted insertional mutagenes is with a kanamycin cassette. Enzymatic assays and reverse transcription PC R carried out with the wild type and with glgP and glgB mutants, as well as primer extension experiments and P-galactosidase fusions, revealed that th is region containing five open reading frames (glgPBCA and pgm) is transcri bed unidirectionally as a single operon under the control of a promoter loc ated upstream of the glycogen phosphorylase gene (glgP). An alternative tra nscript was identified starting 168 bp upstream of an internal ATG start co don of the pgm gene, which is translated as a 71-amino-acid-shorter Pgm pro tein which complements in vivo a pgm mutant. This alternative transcript ha s a promoter with the moth TATCAAN(5)G, identified in octopine Ti plasmid a s an autoinducible TraR promoter. This promoter is >200 times more efficien t in A. tumefaciens than in Escherichia coli, as judged by the level of enz ymatic activity of a lacZ-pgm fusion.