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

Abstract

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. The nucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase (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 (glgP) and branching enzyme (glgB) genes are located immediately upstream of this region. The complete nucleotide sequences of the glgP and glgB genes were obtained, and mutants were constructed by targeted insertional mutagenesis with a kanamycin cassette. Enzymatic assays and reverse transcription PCR carried out with the wild type and with glgP and glgB mutants, as well as primer extension experiments and beta-galactosidase fusions, revealed that this region containing five open reading frames (glgPBCA and pgm) is transcribed unidirectionally as 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 the pgm gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a pgm mutant. This alternative transcript has a promoter with the motif TATCAAN5G, identified in octopine Ti plasmid as an autoinducible TraR promoter. This promoter is >200 times more efficient in A. tumefaciens than in Escherichia coli, as judged by the level of enzymatic activity of a lacZ-pgm fusion.

FIG. 1

Scheme of A. tumefaciens glg operon organization and transcription. The genomic arrangement with the positions of the kanamycin cassette insertions and the putative alternative pgm promoter are shown in the upper part. Oligonucleotides used for RT-PCRs and the primer extension experiment are indicated by arrows. The DNA regions used for the construction of β-galactosidase fusion plasmids pFus98 and pFus97 are shown. The transcriptional organization of the operon, indicating the polycistronic mRNA and the alternative pgm2 mRNA (different scale), is shown in the lower part of the figure. RBS, Shine-Dalgarno conserved sequences; ATG₁ and ATG₂, initiation codons of Pgm1 and Pgm2, respectively.

FIG. 2

RT-PCRs carried out with total RNA obtained from wild-type A348 and A1120 (glgB) mutant strains. (A) Reactions carried out with total RNA of wild-type strain A348. Lane 1, glgP-glgB intergenic region; lane 3, glgB-glgC intergenic region; lane 5, glgC-glgA intergenic region; lane 7, glgA-pgm intergenic region; lane 9, internal region of pgm gene. Lanes 2, 4, 6, 8, and 10 represent control reactions carried out without the addition of reverse transcriptase. Lane a, molecular size markers. (B) Reactions carried out with total RNA of strain A1120 (glgB mutant). Lane 1, glgP-glgB intergenic region; lane 2, internal region of pgm gene. Lane a, molecular size marker. All the other intergenic regions gave negative results (data not shown).

FIG. 3

Mapping of the 5′ end of the pgm gene by primer extension analysis. The transcription start site is indicated by the arrow. Total RNA of the A. tumefaciens A348 wild-type strain was used to synthesize cDNA by employing avian myeloblastosis virus reverse transcriptase and the oligonucleotide PGMPE labeled with [γ-³²P]ATP. The products were subjected to polyacrylamide gel electrophoresis in parallel with the sequencing ladder.

FIG. 4

Nucleotide sequence of the glg gene cluster. Only regions that contained the putative promoters and transcriptional and translational start sites of the 9,100-bp DNA are shown. The sequence from positions 7716 to 7916 has been reported previously (35). Putative ribosome binding sites (RBS) are underlined, start codons are shown with an angled arrow, stop codons are shown with an asterisk, the transcriptional start site is shown with a dot, and putative promoters are in bold type. The complete sequence has been deposited in EMBL GenBank.

FIG. 5

Western blot analysis. Cell extracts were subjected to polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes as described in Materials and Methods. (A) Protein stained with Ponceau S; (B) Western blot carried out with rabbit polyclonal antibody raised against recombinant A. tumefaciens Pgm developed with peroxidase-conjugated antibody against rabbit immunoglobulin G (Dako). Lanes 1, commercial rabbit Pgm; lanes 2, A. tumefaciens wild-type extract; lanes 3, A. tumefaciens pgm mutant A5129 extract; lanes 4, A. tumefaciens glgB mutant A1120 extract. M, prestained molecular mass standards. Arrows on the right indicate the positions of Pgm proteins.