Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1

Abstract

In common with other plant symbionts, Frankia spp., the actinomycete N2-fixing symbionts of certain nonleguminous woody plants, synthesize two glutamine synthetases, GSI and GSII. DNA encoding the Bradyrhizobium japonicum gene for GSII (glnII) hybridized to DNA from three Frankia strains. B. japonicum glnII was used as a probe to clone the glnII gene from a size-selected KpnI library of Frankia strain CpI1 DNA. The region corresponding to the Frankia sp. strain CpI1 glnII gene was sequenced, and the amino acid sequence was compared with that of the GS gene from the pea and glnII from B. japonicum. The Frankia glnII gene product has a high degree of similarity with both GSII from B. japonicum and GS from pea, although the sequence was about equally similar to both the bacterial and eucaryotic proteins. The Frankia glnII gene was also capable of complementing an Escherichia coli delta glnA mutant when transcribed from the vector lac promoter, but not when transcribed from the Frankia promoter. GSII produced in E. coli was heat labile, like the enzyme produced in Frankia sp. strain CpI1 but unlike the wild-type E. coli enzyme.

Identification and cloning of the glnR locus, which is required for transcription of the glnA gene in Streptomyces coelicolor A3(2)

“…species (10,14,38,39,43). In contrast to the GSI enzyme activity, the activity of the GSII enzyme is thermolabile and is not regulated posttranslationally by adenylylation.…”

Purification, Characterization, and Expression of Multiple Glutamine Synthetases from Prevotella ruminicola 23

“…The regulation and structure of typical GSI in enterobacteria have been well studied (1,16,29). GSII, encoded by glnII, is commonly isolated from plant symbiotic bacteria such as Rhizobium japonicum, Frankia sp., Agrobacterium, and Streptomyces hygroscopicus (8,12,19,28,37). GSII is an octameric enzyme with identical subunits (M r , ca.…”

Frankia sequences exhibiting RNA polymerase promoter activity The GenBank accession number for the sequence reported in this paper is AY008259.

“…5a). The glnII gene of Frankia strain CpI1 contains a striking homology to GLO7p1 in a region tentatively identified as encoding the N terminus of the protein (Rochefort & Benson, 1990). An alignment of the Frankia sequences reveals two regions of complete identity : a k35 element, GAGTT, and a k10 region, GATCG (Fig.…”

“…The GLO7 sequence was searched for similarity to promoter elements identified in other bacteria. A sequence similar to the NtrC consensus binding site identified in E. coli and Salmonella (Buck et al, 1986) and to a similar element upstream of the Frankia CpI1 glnII gene (Rochefort & Benson, 1990) was found beginning 101 bp upstream of TSP-1 (Fig. 5b).…”

“…Only a few Frankia genes that encode proteins have been cloned and completely sequenced. These include genes from the nif operon of several strains (Harriott et al, 1995 ;Nalin et al, 1995 ;Normand et al, 1988), ferredoxin subunits from strain EUIK1 (Yoo et al, 1999) and glnA (Hosted et al, 1993) plus glnII (Rochefort & Benson, 1990) from strain CpI1. The rRNA operon of strain ORS020606 (Normand et al, 1992) and partial 16S rRNA sequences have been characterized from many strains (Nazaret et al, 1991 ;Normand et al, 1996).…”