Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli

Abstract

Escherichia coli rpoN mutants lack or4 and are therefore unable to initiate the transcription of ginA at glnAp2, which is required for the production of a high intracellular concentration of glutamine synthetase. We have found that the dependence on a54 can be overcome by iutations that have apparently created a new r70-dependent promoter. The position -35 RNA polymerase contact site of this new promoter overlaps glnAp2.The initiation of transcription at the new promoter is inhibited by r54-RNA polymerase even in the absence of nitrogen regulator I-phosphate, the activator required for the initiation of transcription at glnAp2. The results suggest that in cells growing with an excess of nitrogen and therefore lacking nitrogen regulator I-phosphate, V54-RNA polymergse is bound at glnAp2.Glutamine synthetase is essential for the assimilation of ammonia when Escherichia coli and other species of enteric bacteria are grown in a nitrogen-limited medium. The level of glutamine synthetase and the level of proteins that facilitate the transport and degradation of nitrogen-containing compounds (Ntr systems, nitrogen regulated) are elevated during nitrogen-limited growth (8). The transcription of Ntr genes requires RNA polymerase containing the r54 protein, the product of rpoN (glnF ntrA) rather than the abundant cro protein. We have previously referred to o5 as u6O; however, it has been shown that the molecular weight of this protein based on the amino acid sequence predicted by the nucleotide sequence of rpoN is actually 54,000 (11). The promoters at which transcription is initiated by Cr54 RNA polymerase have the consensus sequence CTGGYAYRN4TTGCA (1,6,7,19).The transcription at these promoters is initiated when the cells experience nitrogen deprivation. The agents responsible for the initiation of transcription are the proteins nitrogen regulators I and II (NRI and NRII), the products, respectively, of glnG (ntrC) and glnL (ntrB). NRII is a kinase responsible for the conversion of NRI to NRI-phosphate, the actual activator of transcription (6,7,12,18). The phosphorylation of NRI is blocked by PI,, the product of glnB. Consequently, transcription requires the removal of PI,. This removal consists in its conversion to the uridylylated form P11-UMP. This reaction is catalyzed by uridylyl transferase, the product of gInD, activated by 2-ketoglutarate. Conversely, the same enzyme is activated by glutamine to remove the uridylyl group from P11-UMP (8,12). As a result of these interactions, the intracellular concentration of PI, and consequently the intracellular concentration of NRIphosphate, the transcriptional activator, is regulated by the intracellular ratio of glutamine to 2-ketoglutarate, a sensitive barometer of the availability of nitrogen. A high intracellular 2-ketoglutarate/glutamine ratio indicating nitrogen defi-* Corresponding author. ciency results in the initiation of transcription at Ntr promoters; a high intracellular glutamine/2-ketoglutarate ratio indicating nitrogen excess prevents the initiation of tra...

Overexpression and Purification of Asparagine Synthetase fromEscherichia coli

“…The active fraction was eluted at a KCl concentration of approximately 0.25 M, and concentrated to about 10 ml on a Amicon UM-10 membrane. The concentrated enzyme was dialyzed against buffer C (the buffer B containing 10mM (CH 3 COOhMg), and then applied to a Blue-Cellulofine column (2.5 x 20 cm) equilibrated with buffer C. The.column was washed with 0.1 M KCl in buffer C, and the enzyme was then eluted with a linear gradient from 0.1 MKCl, 10 mM (CH 3 COOhMg to 1.0 M KCl, 0 mM (CH 3 COOhMg in buffer C. The active fraction was concentrated on the Amicon membrane and dialyzed against buffer B. The puriffed enzyme could be stored at -70°C without loss of activity for at least one month.…”

“…A nucleotide sequence derived from pKK223-3 was underlined with a double line. (CH 3 COOhMg, 1mM EDTA, 30mM KCl, and 5mM 2-mercaptoethanol) and re-collected by centrifugation. The cells were then suspended in 50 ml of buffer A and disrupted by sonication at O°C.…”

“…1 ) These enzymes are distributed in prokaryotes as Escherichia coli 2 ) and Klebsiella aerogenes 3 ) and encoded by asnA genes. On the other hand, asparagine synthetases from eukaryotes 4 -7) use glutamine as the preferred amido nitrogen donor, although they can also use ammonia.…”

Crystallization and preliminary crystallographic study of asparagine synthetase fromEscherichia coli

“…On the other hand, asparagine synthetase using glutamine as amido nitrogen donor [Laspartate: L-glutamine amido-ligase (AMP-forming) E.C. 6.3.5.4] is found not only in prokaryotes (Humbert & Simoni, 1980;Reitzer & Magasanik, 1982) but also in eukaryotes (Andrulis, Chen & Ray, 1987;Gantt & Arfin, 1981;Hongo & Sato, 1981 ;, and encoded by the asnB gene. Studies of steady-state kinetic mechanisms for both enzymes have demonstrated that the mechanism of the ammonia-dependent enzymes is different from that of the glutamine-dependent enzymes (Cedar & Schwartz, 1969b;Hongo & Sato, 1985;Mehlhaff, Luehr & Schuster, 1985).…”

“…6.3.1.1] catalyzes the synthesis of L-asparagine from L-aspartate and ammonia, with hydrolyzing ATP to AMP and PPi (Meister, 1974). This enzyme is distributed in prokaryotes as Escherichia coli (Cedar & Schwartz, 1969a) and Klebsiella aerogenes (Reitzer & Magasanik, 1982), and encoded by the asnA gene. On the other hand, asparagine synthetase using glutamine as amido nitrogen donor [Laspartate: L-glutamine amido-ligase (AMP-forming) E.C.…”