Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product

Abstract

In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product.

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…The gene efg (nadE) was found to code for an ammonium-dependent NAD synthetase (64). Purified NAD synthetase is homodimer with a subunit molecular mass of 30.6 kDa (65). There is strong evidence that ammonium is the sole physiological donor of the amino group for NadE in E. coli and S. Typhimurium (64,66,67), which is in contrast to the eukaryotic enzymes, which are glutamine dependent.…”

“…With the vesicles, binding of glutamine was insensitive to variations in ionic strength and pH (22,65,154,327) and required neither potassium nor phosphate ions, whereas glutamine transport activity was inhibited by increasing ionic strength, had a narrow pH optimum, and required potassium and phosphate ions (328). Glutamine uptake into cells was inhibited by osmotic stress produced by NaCl but was stimulated when sucrose was used instead (329).…”

Ammonia-dependent growth (Adg-) mutants of Rhodobacter capsulatus and Rhodobacter sphaeroides: comparison of mutant phenotypes and cloning of the wild-type (adg A) genes

“…The R . capsulatus mutants RC27, RC28, RC29 and RC34, derived from the wild-type strain B10, showed similar growth properties, except that growth on glutamine was very slow (Willison et al, 1985;Allibert et al, 1987). Strain B10 is unable to utilize NO, as sole nitrogen source, so no conclusion can be drawn from these mutants as to the effects of adgA mutations on NO, assimilation in R .…”

“…pneumoniae. An E. coli gene which complements the 'Ntr-like' mutants of R. capsulatus has been isolated (Allibert et al, 1987) and nucleotide sequencing of the gene has shown it to be unrelated to the known ntr genes of E. coli. The approximate map position of the gene (34-39 min) was determined, and no gene involved in nitrogen metabolism has so far been mapped to this region.…”

Functional analysis of the outB gene of Bacillus subtilis

“…It may be relevant that the E. coli protein homologous to OutB has been shown to be a dimer of M,. 60000 (Allibert et al, 1987).…”