An alternative P_II protein in the regulation of glutamine synthetase in Escherichia coli

Abstract

SummaryThe P II protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that P II , encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB, which encodes a homologue of P II and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as P II , reacted with polyclonal P II antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli P II . Like P II , purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD-dependent fashion. Unlike P II , however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a r N (r 54 ) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/ NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on P II . Possible regulatory implications of this conditional redundancy of P II are discussed.

Product dependence and bifunctionality compromise the ultrasensitivity of signal transduction cascades

“…This protein is obviously crucial for intracellular signal transduction. Yet, it lacked control on the average adenylylation state of GS even in the absence of its paralogue GlnK (55). We found that, especially because the ATase is an ambiguous enzyme, altered expression of total PII protein should not affect the steady-state activity of GS, unless the ratio of PII͞PII-UMP were to change in parallel.…”

“…The authors varied the total concentration of PII. The nonadenylylated GS concentration was completely insensitive to changes in total PII protein level, also when the paralogue of PII, GlnK (54), was absent (55).…”

Modeling the role of covalent enzyme modification inEscherichia colinitrogen metabolism

“…Many authors have pointed out that the bicyclic structure of the cascade serves to integrate signals of the nitrogen (via glutamine) and carbon (via α -KG) status of the cell [11, 25, 30]. Westerhoff and colleagues have proposed [3, 31] that the combination of PII and its homologue GlnK enables the cell to precisely adjust its strategy for GS regulation in response to varying external ammonium concentrations. They find that the combined regulation of GS–GOGAT and GDH allows E. coli to maintain a roughly constant rate of nitrogen influx regardless of ammonium levels [5].…”

Crystallization and preliminary X-ray analysis of Escherichia coli GlnK

“…The DNA sequence encoding GlnK was polymerase chainreaction (PCR) amplified from template plasmid pWVH149 (van Heeswijk et aL, 1996) using a forward primer (5'-AATGGATCCCATATGAA-GCTGGTGACCGTG-3') which introduced a convenient NdeI restriction site (CATATG) at the start and the M13 primer (5'-GTAAAACGCGACGGCCAGT-3'). PCR using standard conditions was carried out with Expand polymerase (Boehringer Mannheim).…”

“…GlnK is a signal-transduction protein which has recently been implicated in the regulation of glutamine synthetase (GS) in Escherichia coli (van Heeswijk et al, 1996). It is a homotrimer with subunits of 112 amino acids and has 67% sequence identity with the Pxi protein, whose role in the regulation of GS in enteric bacteria is well studied (Merrick & Edwards, 1995).…”