Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli

Abstract

SummaryTwo structurally similar but functionally distinct PIIlike proteins, PII and GlnK, regulate nitrogen assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB ) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphor ylation state of the transcriptional activator NRIϳP (NtrCϳP, the phosphorylated form of the glnG (ntrC ) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal-transducing uridylyltransferase/ uridylyl-removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA , which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on nitrogen-rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK *, were obtained as suppressors of the Ntr ¹ phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRIϳP during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

The signal transducer PII and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation a aThe GenBank accession number for the glnB gene sequence reported in this paper is AJ271089.

“…P II and GlnK are structurally similar, but in part functionally distinct proteins (Atkinson & Ninfa, 1998). A P II -like protein has also been identified in the chloroplast genome of the red alga Porphyra purpurea (Reith & Munholland, 1993) and of higher plants such as Arabidopsis thaliana and Ricinus communis (Hsieh et al, 1998).…”

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

“…Although glnB is constitutively expressed in the cell and glnK is only expressed under N-limiting conditions, these two P II homologs have some similar properties (4,5). Both proteins are subject to reversible uridylylation by the bifunctional uridylyltransferase͞uridylyl-removing enzyme (UTase͞UR, gene product of glnD), which is believed to be a primary sensor of intracellular nitrogen status, although curiously GlnK-UMP (the uridylylated form of Glnk) is only poorly deuridylylated by GlnD (6).…”

“…Both proteins are subject to reversible uridylylation by the bifunctional uridylyltransferase͞uridylyl-removing enzyme (UTase͞UR, gene product of glnD), which is believed to be a primary sensor of intracellular nitrogen status, although curiously GlnK-UMP (the uridylylated form of Glnk) is only poorly deuridylylated by GlnD (6). Both proteins can also interact with NtrB to regulate NtrC activity, and with adenylyltransferase (ATase, the glnE product) to modify glutamine synthetase (GS) (4)(5)(6). However, they also display some distinct properties, such as the ability of GlnB-UMP, but not GlnK-UMP, to strongly stimulate the deadenylylation of GS-AMP (the adenylylated form of GS) (7).…”

Two Residues in the T-loop of GlnK Determine NifL-dependent Nitrogen Control of nif Gene Expression

“…coli GlnK can substitute for GlnB functions both in vivo and in vitro although the proteins are not completely interchangeable in all circumstances. In the absence of GlnB, GlnK can regulate adenylylation of GS in vivo and can substitute for GlnB in NtrC-dependent regulation of glnA expression (3,7). Likewise in vitro studies show that both GlnB and GlnK can activate the phosphatase activity of NtrB and the adenylylation of GS by ATase, although GlnK is less effective than GlnB in the latter reaction (4).…”

“…The distinction between the roles of GlnB and GlnK is confounded still further by the recognition that the proteins can form heterotrimers in vivo (8,9). Hence there are clearly still a number of unexplained properties of these proteins, and the precise physiological roles of GlnB and GlnK in E. coli remain to be determined (7,10). There is, however, one well defined function where GlnK has been shown to have a specific role, namely in regulating the interaction between the K. pneumoniae nitrogen fixation regulatory proteins NifL and NifA in response to the intracellular nitrogen status (1,11).…”