Involvement of the product of the glnF gene in the autogenous regulation of glutamine synthetase formation in Klebsiella aerogenes

Abstract

Mutations in a site, glnF, linked by Pl-mediated transduction to argG on the chromosome of Klebsiella aerogenes, result in a requirement for glutamine. Mutants in this gene have in all media a level of glutamine synthetase (GS) corresponding to the level found in the wild-type strain grown in the medium producing the strongest repression of GS. The adenylylation and deadenylylation of GS in glnF mutants is normal. The glutamine requirement of glnF mutants could be suppressed by mutations in the structural gene for GS, ginA. These mutations result in altered regulation of GS synthesis, regardless of the presence or absence of the glnF mutation (GlnR phenotype). In GlnR mutants the GS level is higher than in the wild-type strain when the cells are cultured in strongly repressing medium, but lower than in the wild-type strain when the cells are cultured in a derepressing medium. Heterozygous merodiploids carrying a normal ginA gene as well as a ginA gene responsible for the GhnR phenotype behave in every respect like merodiploids carrying two normal ginA genes. These results confirm autogenous regulation of GS synthesis and indicate that GS is both a repressor and an activator of GS synthesis. The mutation in ginA responsible for the GlnR phenotype has apparently resulted in the formation of a GS that is incompetent both as repressor and as activator of GS synthesis. According to this hypothesis, the product of the glnF gene is necessary for activation of the ginA gene by GS. In enteric bacteria, glutamine synthetase (GS)is reversibly modified by the covalent attachment of an adenylyl moiety to each subunit of the enzyme, thereby rendering it inactive (8). The rate of synthesis of GS in Escherichia coli and Klebsiella aerogenes has been shown to be inversely related to the adenylylation state of GS (16), which in turn depends on the ratio of 2ketoglutarate to glutamine, a very sensitive signal for the availability or lack of ammonia (8,16). If this ratio is high, GS will be derepressed and mostly nonadenylylated; if it is low, GS will be repressed and adenylylated, i.e., biosynthetically inactive.Previous studies in K. aerogenes have suggested the hypothesis that these two means of regulating GS are related: adenylylated GS might repress the synthesis of GS or non-adenylylated GS might activate it (6). The properties of four different types of mutants support this view. Strains carrying mutations at either the ginB or ginD locus (6, 12) appear to be unable to deadenylylate GS, and accordingly contain low levels of GS, mostly in adenylylated form. t Present address:

Positive and negative control of the glnA ntrBC regulon in Klebsiella pneumoniae.

“…Mutations in ntrC suppress the glutamine requirement of ntrA mutants and the resultant double mutants have the low GS values characteristic of ntrC mutants (Gaillardin and Magasanik, 1978;Pahel and Tyler, 1979;Kustu et al, 1979;Espin et al, 1982). We suggest that the low expression of ginA observed in ntrC mutants is due to the ntrBCindependent activity of P1 observed in our experiments.…”

“…In all cases it comprises three genes: ntrA (ginF), ntrB (glnL) and ntrC (ginG). The ntrBC (glnLG) genes are linked to the structural gene for glutamine synthetase (GS) ginA (McFarland et al, 1981;Espin et al, 1982; Goldie and Magasanik, 1982) whereas ntrA (glnF) is unlinked (Garcia et al, 1977;Leonardo and Goldberg, 1980;Pahel et al, 1978;Gaillardin and Magasanik, 1978). In E. coli the ginA and ntrBC (glnLG) genes form a comlex operon P1 glnA P2 ntrBC (glnLG), in which, under conditions of nitrogen limitation, ntrB (glnL) and ntrC (glnG) are transcribed predominantly from the P1 promoter, whilst in N-excess transcription from P1 is repressed and ntrBC (glnLG) expression is predomi-IRL Press Limited, Oxford, England.…”

The RpoN‐box motif of the RNA polymerase sigma factor σN plays a role in promoter recognition

“…Growth on nitrogen sources. RpoN (glnF ) was originally identified in S. typhimurium and E. coli by the isolation of a novel class of Gln auxotrophs (Garcia et al, 1977;Gaillardin and Magasanik, 1978). However, mutations in rpoN are surprisingly rare amongst Gln auxotrophs in E. coli (MacNeil et al, 1982) and most of the previously isolated rpoN point mutations in K. pneumoniae do not cause Gln auxotrophy whereas an rpoN deletion (⌬rpoN71::kan) is Gln ¹ (M. Merrick, unpublished).…”

“…However, mutations in rpoN are surprisingly rare amongst Gln auxotrophs in E. coli (MacNeil et al, 1982) and most of the previously isolated rpoN point mutations in K. pneumoniae do not cause Gln auxotrophy whereas an rpoN deletion (⌬rpoN71::kan) is Gln ¹ (M. Merrick, unpublished). A further characteristic phenotype of rpoN mutants is their failure to utilize a range of nitrogen sources (Gaillardin and Magasanik, 1978;Coppard and Merrick, 1991) and the growth phenotypes of each of the mutants on Arg or His as the sole N source was therefore tested by introduction of each rpoN allele on a low-copy-number plasmid into the rpoN deletion strain UNF2792. Of the 12 mutants analysed, four grew poorly on His and Arg (Ntr ¹ phenotype) but only two of these showed significant Gln auxotrophy ( Table 2).…”

Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine

“…In Escherichia coli and other Gram-negative microorganisms tested, the regulation of GS occurs by a variety of mechanisms: the enzymatic adenylylation of one tyrosine residue in each subunit of the enzyme, the cumulative feedback inhibition by several metabolites, and the interconversion of active (taut) and inactive (relaxed) forms of the enzyme in response to fluctuations in the concentration of divalent cations (for reviews, see Ginsburg, 1974 andStadtman et al 1979). Moreover, a model of autogenous regulation has been proposed in which GS regulates its own synthesis (Foor et al, 1975;Streicher et al, 1975;Bender and Magasanik, 1977; Gaillardin and Magasanik, 1978). Finally, a correlation can be made between the percentage of charged tRNAglutamate and the level of glutamine synthetase in a thermosensitive mutant of Escherichia coli altered in the glutamyl-tRNA synthetase (Lapointe et al, 1975), suggesting that the biosynthesis of GS might also be regulated at the level of the at-tenuation of the transcription of its structural gene, as has been found for many bacterial operons concerned with the biosynthesis of amino acids (for a review, see Yanofsky, 1981).…”