Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes nfe.

Rhizobium meliloti large plasmid pRmeGR4b carries the nodulation competitiveness locus nfe responsible for the nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. We report here the nucleotide sequence and characterization of a 3345 base-pair DNA section of the nfe region. Sequence analysis revealed four open reading frames (ORFs), two of them with rightward polarity, termed nfe1 and nfe2, are preceded by functional nif consensus sequences and NifA-binding motifs. An additional, NifA-independent, transcriptional start site for nfe1 was also found. Two other ORFs with leftward polarity, designated ORFA and ORFB, were identified upstream from nfe1 and nfe2 but no nif consensus sequences were found. However, expression of ORFA might be indirectly coupled to the NifA-NtrA regulatory network. The gene products of nfe1 and nfe2 were identified using in vitro transcription/translation and bacteriophage T7 RNA polymerase/promoter system, respectively. A high degree of homology between the amino terminal domain of Nfe1 and the nifH gene product was found. In addition, nfe1 shows homology with the upstream non-coding DNA region of the fixABCX operon. Furthermore, the putative ORFB encoded protein contains a helix-turn-helix motif that resembles the DNA-binding consensus sequence proposed for many prokaryotic regulatory proteins.

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 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.

Further studies on the "de novo" process of alpha 1,4-alpha 1,6 glucopolysaccharides--corn starch biogenesis.

Starch biogenesis in corn endosperm from Flint, Sugary, Waxy, as a function of the grain filling/period was studied. We have differentially identified the initiation from the elongation process. After incubating under unprimed conditions, two glucose radiolabelled protein bands of 39,5 and 36 kDa were obtained. UDP(14C)Glc was the preferred glucosyl donor but also ADP(14C)Glc was. It was additionally found that more than one glucose was transferred to the protein or to the alpha 1,4-glucan linked to protein from UDPGlc. These results were supported by the fact that the glucosylated protein from UDPGlc liberates maltooligosaccharides after alpha- or beta-amylase treatment. The elongation activity in the first steps related to the glucan linked to protein is different from starch synthase. Therefore, we are proposing a model for starch biogenesis where two new transglucosylating enzyme activities are necessary to prepare the primer for starch synthase.