L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase.

An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression.     

Autogenous regulation of the synthesis of glutamine synthetase in Klebsiella aerogenes.

We isolated an F' episome of Escherichia coli carrying the glnA+ gene from K. aerogenes and an F' episome of E. coli carrying the glnA4 allele from K. aerogenes responsible for the constitutive synthesis of glutamine synthetase. Complementation tests with these episomes showed that the glnA4 mutation (leading to the constitutive synthesis of active glutamine synthetase) was in the gene identified by mutations glnA20, glnA51, and glnA5 as the structural gene for glutamine synthetase. By using these merodiploid strains we were able to show that the glnA51 mutation lead to the synthesis of a glutamine synthetase that lacked enzymatic activity but fully retained its regulatory properties. Finally, we discuss a model that explains the several phenotypes associated with mutations such as glnA4 located within the structural gene for glutamine synthetase leading to constitutive synthesis of active glutamine synthetase.     

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.     

Isolation of Klebsiella aerogenes mutants cis-dominant for glutamine synthetase expression

We have isolated three strains of Klebsiella aerogenes that failed to show repression of glutamine synthetase even when grown under the most repressing conditions for the wild-type strain. These mutant strains were selected as glutamine-independent derivatives of a strain that is merodiploid for the glnA region and contains a mutated glnF allele. The mutation responsible for the Gln+ phenotype in each strain was tightly linked to glnA, the structural gene for glutamine synthetase, and was dominant to the wild-type allele. These mutations are probably lesions in the control region of the glnA gene, since each mutation was cis-dominant for constitutive expression of the enzyme in hybrid merodiploid strains. Strains harboring this class of mutations were unable to produce a high level of glutamine synthetase unless they also contained an intact glnF gene, and unless cells were grown in derepressing medium. This study supports the idea that the glnA gene is regulated both positively and negatively, and that the deoxyribonucleic acid sites critical for positive control and negative control are functionally distinct.     

Genetic control of glutamine synthetase in Klebiella aerogenes.

Mutations at two sites, glnA and glnB, of the Klebsiella aerogenes chromosome result in the loss of glutamine synthetase. The locations of these sites on the chromosome were established by complementation by episomes of Escherichia coli and by determination of their linkage to other genetic sites by transduction with phage P1. The glnB gene is located at a position corresponding to 48 min on the Taylor map of the E. coli chromosome; it is linked to tryA, nadB, and GUA. The glnA gene is at a position corresponding to 77 min on the Taylor map and is linked to rha and metB; it is also closely linked to rbs, located in E. coli at 74 min, indicating a difference in this chromosomal region between E. coli and K. aerogenes. Mutations in the glnA site can also lead to nonrepressible synthesis of active glutamine synthetase. The examination of the fine genetic structure of glnA revealed that one such mutation is located between two mutations leading to the loss of enzymatic activity. This result, together with evidence that the structural gene for glutamine synthetase is at glnA, suggests that glutamine synthetase controls expression of its own structural gene by repression.     

Identification of the structural gene for glutamine synthetase in Klebsiella aerogenes.

Mutations at two sites of the Klebsiella aerogenes chromosome, unlinked by transduction with phages PW52 and P1, result in the lack of enzymatically active glutamine synthetase. A mutation in the glnB site leads to a marked decrease in the formation of an apparently normal enzyme. Some of the mutations in the glnA site lead to the production of enzymatically inactive material capable of reacting with anti-glutamine synthetase serum. The revertant of a glnA mutant was found to produce a glutamine synthetase with less activity and less stability to heat than the enzyme of the wild type. These results locate the structural gene to the production of enzymatically inactive glutamine synthetase antigen, not subject to repression by exogenously added ammonia. This observation suggests that glutamine synthetase is itself involved in the regulation of the synthesis of glutamine synthetase.     

Catabolic N2-acetylornithine 5-aminotransferase of Klebsiella aerogenes: control of synthesis by induction, catabolite repression, and activation by glutamine synthetase.

Klebsiella aerogenes formed two N2-acetylornithine 5-aminotransferases (ACOAT) which were separable by diethylaminoethyl-cellulose chromatography. One ACOAT was repressed when the cells grew on arginine-containing medium, indicating its function in arginine biosynthesis. The second ACOAT was induced when arginine or ornithine was present in the medium as the sole source of carbon or nitrogen, suggesting its function in the catabolism of these compounds. The induced enzyme was purified almost to homogeneity. Its molecular weight is 59,000; it is a pyridoxal 5-phosphate-dependent enzyme and exhibits activity with N2-acetylornithine (Km = 1.1 mM) as well as with ornithine (Km = 5.4 mM). ACOAT did not catalyze the transamination of putrescine or 4-aminobutyrate. The best amino acceptor was 2-ketoglutarate (Km = 0.7 mM). ACOAT formation was subject to catabolite repression exerted by glucose when ammonia was present in excess. When the cells were deprived of nitrogen, ACOAT escaped from catabolite repression. This activation was mediated by glutamine synthetase as shown by the fact that mutants affected in the regulation or synthesis of glutamine synthetase were also affected in the control of ACOAT formation.     

Regulatory mutations in the Klebsiella aerogenes structural gene for glutamine synthetase.

Glutamine synthetase could be repressed several hundredfold rather than 6- to 10-fold as previously reported. Ammonia was not the primary repression signal for glutamine synthetase. Repression appeared to be mediated by a high level of glutamine and probably by a high ratio of glutamine to alpha-ketoglutarate. Mutations in glnA (the structural gene for glutamine synthetase) were seen to fall into three phenotypic groups: glutamine auxotrophs that produced no detectable glnA product; glutamine auxotrophs that produced a glnA product lacking enzymatic activity (and hence repressibility by ammonia) but were repressible under appropriate conditions; and glutamine synthetase regulatory mutants, whose glnA product was enzymatically active and not repressible under any conditions.     

Regulation of enzyme formation in Klebsiella aerogenes by episomal glutamine synthetase of Escherichia coli.

We studied the physiology of cells of Klebsiella aerogenes containing the structural gene for glutamine synthetase (glnA) of Escherichia coli on an episome. The E. coli glutamine synthetase functioned in cells of K. aerogenes in a manner similar to that of the K. aerogenes enzyme: it allowed the level of histidase to increase and that of glutamate dehydrogenase to decrease during nitrogen-limited growth. The phenotype of mutations in the glnA site was restored to normal by the introduction of the episomal glnA+ gene. These results are consistent with the hypothesis that glutamine synthetase regulates the function of its own structural gene.     

Regulation of glutamine synthetase by regulatory protein PII in Klebsiella aerogenes mutants lacking adenylyltransferase.

A mutation of Klebsiella aerogenes causing production of an altered PII regulatory protein which stimulates overadenylylation of glutamine synthetase and also prevents its derepression was combined with mutations abolishing the activity of adenylyltransferase. The results support the idea that PII plays a role in the regulation of the level of glutamine synthetase which is independent of its interaction with adenylyltransferase.     

Genetic control of arylsulfatase synthesis in Klebsiella aerogenes.

It was shown that at least four genes are specifically responsible for arylsulfatase synthesis in Klebsiella aerogenes. Mutations at chromosome site atsA result in enzymatically inactive arylsulfatase. Mutants showing constitutive synthesis of arylsulfatase (atsR) were isolated by using inorganic sulfate or cysteine as the sulfur source. Another mutation in which repression of arylsulfatase by inorganic sulfate or cysteine could not be relieved by tyramine was determined by genetic analysis to be on the tyramine oxidase gene (tyn). This site was distinguished from the atsC mutation site, which is probably concerned with the action or synthesis of corepressors of arylsulfatase synthesis. Genetic analysis with transducing phage PW52 showed that the order of mutation sites was atsC-atsR-atsA-tynA-tynB. On the basis of these results and previous physiological findings, we propose a new model for regulation of arylsulfatase synthesis.     

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a 'marking' step in protein turnover.     

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.     

Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase.

The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA.     

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

Mutations in a site, glnF, linked by P1-mediated transduction of 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, glnA. 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 cells are cultured in a derepressing medium. Heterozygous merodiploids carrying a normal glnA gene as well as a glnA gene responsible for the GlnR phenotype behave in every respect like merodiploids carrying two normal glnA 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 glnA 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 glnA gene by GS.     

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation.     

Serine utilization by Klebsiella aerogenes.

Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions.     

Enzymes of agmatine degradation and the control of their synthesis in Klebsiella aerogenes.

The degradation of agmatine to succinate by Klebsiella aerogenes occurs in five steps. The enzyme catalyzing the first step, agmatinase, is induced by agmatine. The enzymes catalyzing the second and third steps, putrescine aminotransferase and 4-aminobutyraldehyde dehydrogenase, are induced by putrescine and also by their product, 4-aminobutyrate. The enzymes catalyzing the fourth and fifth steps, 4-aminobutyrate aminotransferase and succinate semialdehyde dehydrogenase, are induced by 4-aminobutyrate. This compound also serves as gratuitous inducer of the catabolic acetylornithine aminotransferase. The formation of the enzymes responsible for agmatine degradation is regulated not only by induction, but also by catabolite repression and activation by glutamine synthetase.