Sydney Kustu

Covalent modification of bacterial glutamine synthetase: physiological significance

SummaryStadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH4+). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to ∼10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH4+ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH4+-excess conditions and thereby to allow rapid growth.

Glutamine auxotrophs with mutations in a nitrogen regulatory gene, ntrC, that is near glnA

SummarySome mutations to glutamine auxotrophy in the 86 unit region of the Salmonella chromosome lie within the nitrogen regulatory gene, ntrC, rather than the structural gene encoding glutamine synthetase, glnA. Assignment of mutations to ntrC is based on fine structure mapping by P22-mediated transduction and on complementation analysis. Strains with ntrC lesions that cause glutamine auxotrophy (NtrCrepressor) have very low levels of glutamine synthetase (lower than those of strains that completely lack ntrC function and comparable to those of strains that lack atrA function). NtrCrep strains fail to increase synthesis of glutamine synthetase or several amino acid transport components under nitrogen limiting conditions. Thus, like ntrA strains, they appear to repress glnA transcription and fail to activate transcription of glnA or other nitrogen controlled genes. Mutations that suppress the glutamine requirement caused by NtrCrep lesions arise at high frequency; these mutations also suppress the glutamine requirement caused by ntrA lesions. Several suppressor mutations result in loss of function of ntrC.

Adenylylation of Bacterial Glutamine Synthetase: Physiological Significance

Publisher Summary This chapter focuses on the adenylylation of bacterial glutamine synthetase. The enzyme glutamine synthetase catalyzes the synthesis of glutamine from ammonia and glutamate in an ATP-dependent reaction. In bacteria and higher plants, it also participates in net synthesis of glutamate from ammonia and 2-oxoglutarate by functioning in a cycle with glutamate synthase. Physiological studies of Holzer and colleagues indicated that the GS of Escherichia coli was very rapidly adenylylated when the organism was shifted from N-limited medium to a medium containing excess ammonium. Strains of S. typhimurium (glnE) that lack the ability to adenlylate GS show a large growth defect upon shift from N-limited media, media in which they have accumulated high levels of GS, to media containing a high concentration of NH 4 +. This growth defect appears to be because of excessive GS activity after the shift because it is eliminated in glnE strains that also synthesize abnormally low amounts of GS in both N-limited and NH4+, it is prolonged in glnE strains that also synthesize abnormally high amounts of GS in NH4+ excess media and the latter strains continue to show a growth defect even after adaptation to NH 4 +, and glnE strains have normal amounts of both of the glutamatebiosynthetic enzymes under N-limited conditions and after NH 4 + upshift. At present, there appears to be no simple explanation for the distribution of this covalent modification. Although adenylylation of GS occurs widely among gram-negative bacteria, a more complete understanding of the physiological requirements for adenylylation of GS will contribute to understanding the basis for distribution of this covalent modification among the prokaryotes.

Evidence that nitrogen regulatory gene ntrC of Salmonella typhimurium is transcribed from the glnA promoter as well as from a separate ntr promoter.

SummaryPrevious work has indicated that nitrogen regulatory genes ntrB and ntrC of Salmonella typhimurium are closely linked to glnA, the structural gene encoding glutamine synthetase; proceeding clockwise the order of genes in the 86 U region of the map is polA... ntrC ntrB glnA glnApromoter... rha. To study ntrC transcription we have constructed operon fusions of ntrC to lacZ using the Casadaban Mu d1 (Aprlac) phage so that we can measure β-galactosidase activity as a reflection of ntrC transcription and we have introduced into fusion strains promoter constitutive mutations at glnA [glnAp(Con)]. The glnAp(Con) mutations, which elevate glnA expression in fusion strains, also elevate β-galactosidase activity, indicating that ntrC is cotranscribed with glnA. Consistent with this interpretation, polar insertion mutations in glnA decrease β-galactosidase activity of fusion strains carrying glnAp(Con) mutations. However, glnA insertions do not eliminate β-galactosidase activity of glnAp(Con) ntrC::Mu d1 strains and they have little effect on β-galactosidase activity of the original ntrC::Mu d1 fusion strains. The latter results confirm that ntrC can also be transcribed from an ntr promoter downstream of glnA. Polar insertion mutations in ntrB eliminate β-galactosidase activity of both the original fusion strains and fusion strains carrying glnA(Con) mutations, indicating that the ntr promoter lies between glnA and ntrB.

Regulation of transcription of glnA, the structural gene encoding glutamine synthetase, in glnA::Mu d1 (Apr, lac) fusion strains of Salmonella typhimurium

SummaryUsing the Casadaban Mu d1 phage (Casadaban and Cohen 1979) we fused cis-acting regulatory sites for the Salmonella typhimurium glnA gene, the structural gene encoding glutamine synthetase, to lacZ so that transcription of lacZ was controlled by the glnA promoter-operator. Activities of β-galactosidase in two glnA:: Mu d1 fusion strains were high, approximately 25% and 125% the induced level of β-galactosidase when transcription of lacZ is under control of the lac promoter, indicating that glutamine synthetase is not required to activate transcription of its own structural gene. Introduction of nitrogen regulatory mutations ntrA:: Tn10 or ntrC:: Tn10 into fusion strains resulted in greatly decreased synthesis of β-galactosidase indicating that the positive regulatory factors encoded by ntrA and ntrC activate glnA expression at the level of transcription. Comparison of β-galactosidase activities in fusion strains with those in fusions carrying ntrC or ntrA mutations indicated that: 1) the magnitude of activation of glnA expression is at least 43-fold; 2) the magnitude of repression is approximately 13-fold and repression occurs at the level of transcription; 3) the degree of modulation of glnA expression by ntr products is at least 560-fold (13x43); and 4) glutamine synthetase is not required for repression of transcription of its own structural gene. In contrast to strains carrying non-polar mutations in glnA, strains carrying glnA insertion mutations, including glnA:: Mu d1 fusions, are apparently defective in activating expression of some nitrogen controlled genes other than glnA. Defects cannot be accounted for by the absence of glutamine synthetase protein or catalytic activity; they appear to be due to decreased expression of nitrogen regulatory genes ntrB and/or ntrC, which are adjacent to glnA.

Characterization of mutations that lie in the promoter-regulatory region for glnA, the structural gene encoding glutamine synthetase

SummaryIn enteric bacteria products of nitrogen regulatory genes ntrA, ntrB and ntrC are known to regulate transcription both positively and negatively at glnA, the structural gene encoding glutamine synthetase [L-glutamate: ammonia-ligase (ADP-forming), EC 6.3.1.2]. We have characterized two types of cis-acting mutations in the glnA promoter-regulatory region. One type, which we have called promoter Up [glnAp (Up)], elevates transcription of glnA to high levels without need for ntr-mediated activation but leaves expression sensitive to ntr-mediated repression. The other type renders glnA transcription insensitive to repression but leaves it normally responsive to activation. Properties of the two types of promoter-regulatory mutations suggest that sites for ntr-mediated activation of glnA transcription are functionally distinct from sites for ntr-mediated repression.

Characterization of ?glnA + phages used as templates for in vitro synthesis of glutamine synthetase

SummarySeven λglnA+ transducing phages carrying different amounts of Escherichia coli bacterial DNA to the left of the lambda attachment site could be grouped into two families which apparently originated from two secondary bacterial attachment sites clockwise of glnA. These conclusions are based on analysis of the phages with restriction endonucleases EcoRI and BstI and on analysis of their function in directing protein synthesis in a coupled in vitro transcription-translation system. DNA from all phages directed synthesis of glutamine synthetase, the product of the glnA gene, which was identified immunologically and by subunit molecular weight. All but the shortest phage of one family directed synthesis of the products of nitrogen regulatory genes ntrB and ntrC, which are closely linked to an counter-clockwise of glnA on the bacterial chromosome. Synthesis of glutamine synthetase from several phages in vivo was regulated by availability of nitrogen and required a functional ntrA product, indicating that the glnA promoter-operator was intact.