Gene Organization and Transcription Analysis of Agrobacterium tumefaciens Glycogen (glg) Operon. Two Transcripts for the Single Phosphoglucomutase Gene

J.E. UGALDE, V. LEPEK, A. UTTARO, J. ESTRELLA, A.A. IGLESIAS Y R.A. UGALDE

JOURNAL OF BACTERIOLOGY

American Society for Microbiology

Año: 1998 vol. 180 p. 6557 - 6564

0021-9193

The gene organization and transcription of the Agrobacterium glg operon differ from those in other bacteria.Agrobacterium tumefaciens A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism. Thenucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase (glgC),glycogen synthetase (glgA), and phosphoglucomutase (pgm) genes have been previously described (A. Uttaroand R. A. Ugalde, Gene 150:117–122, 1994). In this work we report that the glycogen phosphorylase (glgP) andbranching enzyme (glgB) genes are located immediately upstream of this region. The complete nucleotidesequences of the glgP and glgB genes were obtained, and mutants were constructed by targeted insertionalmutagenesis with a kanamycin cassette. Enzymatic assays and reverse transcription PCR carried out with thewild type and with glgP and glgB mutants, as well as primer extension experiments and b-galactosidase fusions,revealed that this region containing five open reading frames (glgPBCA and pgm) is transcribed unidirectionallyas a single operon under the control of a promoter located upstream of the glycogen phosphorylase gene(glgP). An alternative transcript was identified starting 168 bp upstream of an internal ATG start codon of thepgm gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a pgmmutant. This alternative transcript has a promoter with the motif TATCAAN5G, identified in octopine Tiplasmid as an autoinducible TraR promoter. This promoter is >200 times more efficient in A. tumefaciens thanin Escherichia coli, as judged by the level of enzymatic activity of a lacZ-pgm fusion.