Regulation of the synthesis of enzymes responsible for glutamate formation in Klebsiella aerogenes.

Abstract A mutant of Klebsiella aerogenes lacking glutamine (amide):α-ketoglutarate (NADP+) amidotransferase oxidoreductase (glutamate synthetase) cannot grow in minimal media containing ammonia as the only nitrogen source at a concentration lower than 1 mm. In addition, in a glucose containing medium, it fails to utilize amino acids, such as histidine, that are converted to glutamate, as sources of nitrogen. It can use glutamate as source of nitrogen. Revertants were isolated capable of growth on glucose-histidine, but unable to use ammonia. These revertants still lack glutamate synthetase, but produce glutamine synthetase constitutively (glnC-). They fail to produce glutamate dehydrogenase, and thus cannot use ammonia as a nitrogen source. An analysis of the levels of glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase in the parent organism, in mutants lacking glutamine synthetase, and in mutants producing it constitutively, reveals that glutamine synthetase represses the formation of glutamate dehydrogenase. A lack of glutamate synthetase appears to interfere with the derepression of glutamine synthetase. On the other hand, the loss of glutamate dehydrogenase by mutation does not affect glutamine synthetase. These results, together with those reported in an earlier paper (Prival, M. J., Brenchley, J. E., and Magasanik, B. (1973) J. Biol. Chem. 248, 4334–4344), indicate that glutamine synthetase is the key element in the regulation of the synthesis of enzymes capable of supplying the cell with glutamate.

It can use glutamate as source of nitrogen. Revertants were isolated capable of growth on glucose-histidine, but unable to use ammonia.
They fail to produce glutamate dehydrogenase, and thus cannot use ammonia as a nitrogen source. An analysis of the levels of glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase in the parent organism, in mutants lacking glutamine synthetase, and in mutants producing it constitutively, reveals that glutamine synthetase represses the formation of glutamate dehydrogenase. A lack of glutamate synthetase appears to interfere with the derepression of glutamine synthetase.
On the other hand, the loss of glutamate dehydrogenase by mutation does not affect glutamine synthetase.
These results, together with those reported in an earlier paper ( We have previously observed a correlation between the level of glutamine synthetase and the ability of Klebsiella aerogenes to produce histidase and proline oxidase in the presence of glucose (2). These enzymes are required for the conversion of histidine * This work was supported by Research Grants GM-07446 and AM-13894 from the National Institutes of Health and Grant GB-32509 from the National Science Foundation.
A preliminary report has been published (lj.
d Present address, Department of Microbiology, Pennsylvania State University, Univeisity Park, Pennsylvania.
6 Present address. Center for Science in the Public Interest, 1179 Church Street,'N.W., Washington, D.C. 1 To whom reprint requests should be sent. and proline, respectively, to glutamate.
We now find that the loss of glutamine (amide) :a+ketoglutarate (NADP+) amidotransferase oxido-reductase (glutamate synthetase) prevents the relief of histidase from catabolite repression that normally occurs during starvation for a source of nitrogen (3). This effect caused by the lack of glutamate synthetase also appears to be mediated through glutamine synthetase.
Glutamine synthetase and glutamate synthetase acting in concert catalyze the ATP-dependent conversion of NH3 and cY-ketoglutarate to glutamate (4 We find that the level of glutamate dehydrogenase is inversely related to the level of glutamine synthetase. Together these observations suggest that glutamine synthetase is a key element in the control of the formation of enzymes able to supply the cell with glutamate.  Table II of an earlier paper (2). Additional strains are listed in Table I. The procedures for mutagenesis have been described (3). Strain MK-19 (pur-1) was isolated from wild type strain MK-1 after mutagenesis with ethylmethanesulfonate.
A culture of MK-19 was treated with ethylmethanesulfonate, washed, and grown in histidine medium.
(All media used in the selection were supplemented with adenine.) A penicillin selection was performed for mutants unable to grow in medium containing glucose with 0.2% urocanate as the sole nitrogen source. Surviving cells (2) a The glnC mutation suppresses the glnB mutation; the phenotype of MK-94 is therefore Gin+, GlnC-(2).
were grown in glucose plus ammonium sulfate medium, followed by growth on histidine to eliminate Hut negative mutants. The resulting culture contained cells able to utilize histidine as sole source of carbon and nitrogen, but unable to utilize urocanate or histidine as a nitrogen source in the presence of glucose. A clone isolated from this culture with these properties was designated MK-164.
This strain was transduced with phage grown on wild type strain MK-1 and a purine-independent transductant was selected and named MK-189.
Spontaneous revertants of MK-189 were selected for the ability to grow well on glucose-histidine medium. Two of these revertants are MK-204 and MK-208.
Strain MK-256, which lacks glutamate synthetase and is constitutive for the synthesis of the Hut enzymes, was isolated as follows.
Strain MK-255 was selected from MK-189 as being unable to grow on histidine after ethylmethanesulfonate mutagenesis and penicillin selection. This strain was transduced with phage grown on hufC515 strain MK-53, and transductants 6123 were selected for their ability to grow on histidine medium. Due to the close linkage of the hulC515 mutation to the histidine utilization mutation in the recipient strain, transductants containing the hutC515 mutation were obtained.
One such strain was named MK-256.
Growth experiments and enzyme assays showed MK-256 to have retained the inability to produce glutamate synthetase and to be constitutive for histidase synthesis.
Mutants lacking glutamate dehydrogenase were isolated by the following procedure. Strain MK-189 was treated with ethylmethanesulfonate and mutants unable to grow without added glutamate were isolated as described previously (3). One of these mutants, strain MK-261, failed to grow not only on glucose, but also on acetate, succinate, or citrate, unless the medium was supplemented with glutamate. Extracts of this mutant, in contrast to those of the parent strain, did not contain glutamate dehydrogenase activity (see Table VI).
The strain, like its parent MK-189, lacks glutamate synthetase (see Table  VI).
Strain MK-261 was treated with a phage lysate of strain MK-1, and transductants capable of growth on glucose-ammonia medium were isolated.
Among the transductants, some had also acquired the ability to grow on glucose-histidine. Extracts of cells of these transductants, such as strain MK-270, were found to contain glutamate synthetase, but not glutamate dehydrogenase activity.

Enzyme
Assays-Whole cell assays as described by Prival and Magasanik (3) were used for histidase and fl-galactosidase determinations.
Enzyme activity was related to the optical density at 420 nm of dilutions of the untreated cell suspension. Cells were sonically disrupted and extracts were prepared for t.he glutamate dehydrogenase, glutamate synthetase, and glutamine synthetase assays (2). The assays for glutamate dehydrogenase and glutamate synthetase are those described by Meers et al. (4). The glutamine synthetase assay was the "transferase" reaction described by Stadtman et al. (5) and in detail in an earlier paper (2).
Protein determinations on extracts were carried out by the method of Lowry et al. (6).
Because assays of mutant MK-189 showed some residual glutamate synthetase activity even though there was no growth on media containing less than 1 mM ammonia (indicating that the mutation was not leaky), we wanted to determine the cause of this activity.
We considered the possibility that the activity measured was in fact not glutamate synthetase, but glutamate dehydrogenase.
This possibility is a likely one, since the assay mixtures for the two enzymes are identical, except that 40 mM NH,Cl replaces 5 mM glutamine when the assay is for glutamate dehydrogenase.
The crude extracts used and the glutamine solution are not entirely free of NHd+.
We used DON,' the kind gift of Dr. S. Hartman of Boston University.
This compound is an analogue to glutamine, known to inhibit the transfer of the amide group of glutamine (7), and should therefore inhibit glutamate synthetase but not glutamate dehydrogenase.
We carried out the assays for these two enzymes, after preincubation of the mixture containing the cell extract with increasing concentration of DON for 10 min before addition of the substrate. The results, summarized in Table II, show that with extracts from strain MK-94, which lacks glutamate dehydrogenase (see Table VI), 0.1 rnM DON completely inhibits glutamate synthetase. With extracts from strain MK-53 where the specific activity of glutamate synthetase is approximately 100 units per mg of protein, the specific activity of glutamate synthetase was 1 The abbreviation used is: DON, 6-diazo-5-oxo-1-norleucine.
apparently reduced by 0.02 to 0.1 m&I DON from 96 to 10 units per mg of protein.
We assume that this residual activity is actually due to glutamate dehydrogenase.
Consequently, we have corrected all reported assays for glutamate synthetase by subtracting 10% of the measured specific activity of glutamate dehydrogenase.
We have also found substantial instability of the glutamate synthetase activity during preparation and with assay conditions described making the observed fluctuations difficult to interpret (8). However, the assay is reliable enough to distinguish the reproducible low activity found in MK-189 extracts from the higher levels found in other strains.

Mutant
with CnB Phenotype--We have presented evidence that mutants of K. aerogenes isolated as glutamine requirers are unable to synthesize glutamine synthetase (Gin-) and are also unable to relieve the catabolite repression of histidase under conditions of nitrogen limitation (CnS) (2). A revertant of one of these glu- tamine requirers overproduces glutamine synthetase under all growth conditions tested (GhlC-), and its histidase is not subject to cat'abolite repression (Cn") (2). We now set out to determine whether a mutant could be isolated which is unable to relieve its catabolite repression of histidase (CnS) during nitrogen limitation but which does not require glutamine (Gin+).
Such a CnS mutant should be unable to use histidine or urocanic acid effectively as a source of nitrogen in the presence of glucose, but should grow in a medium containing histidine as sole carbon source.
The CnS mutant was isolated as described under "Experimental Procedure"; strain 1\IK-189 grows normally on histidine alone, but grows in the glucose-histidine medium with a generation time of 312 min while its parent grows in this medium with a generation time of 78 min (Fig. 1). The regulation of histidase in MK-189 is shown in Table III.
Hist.idase synthesis is induced by histidine and repressed by glucose as it is in wild type strain MK-1.
However, when the supply of nitrogen is limited by growth on hist,idine as sole nitrogen source in the presence of glucose, histidasc synthesis is derepressed in the wild type strain 1\IK-1 but not in the mutant strain MK-189.
One possible explanation for the slow growth of MK-189 on the glucose-histidine medium might be a mutation within the hut region altering the response of the hut enzymes to catabolite repression.
However, transduction experiments failed to reveal any linkage of the CnS phenotype to the hut region making this explanation unlikely. MK-189 and MK-1 were tested for their ability to utilize each of the 20 common amino acids as a source of nitrogen in the presence of glucose. The wild type, ?\IK-1, grows on media containing glucose plus any of the amino acids tested except lysine, isoleucine, or leucine; MK-189 grows well only on glucose medium containing glutamic acid, glutamine, aspartic acid, or asparagine as sole source of nitrogen.
Growth of the mutant on glucose plus other amino acids is significantly slower than that of the wild type (Table IV).
When MK-189 was transduced with a phage lysate grown on MK-1, transductants selected for their ability to grow on the glucose-histidine medium were found to  The experiments were carried out as described in Table  III  except that the media contained 1 mM isopropyl thio-@-n-galactoside.
The enzyme levels are given as nanomoles of product formed per min per ml of cell suspension with an optical density of 1.0 at 420 nm. we changed the control of its hut system from inducible to constitutive. This was accomplished as described under "Experimental Procedure," by isolating a Hut-mutant of MK-189 and transducing it to Hut+ with phage grown on a strain MK-53 (hutC515).
One such transductant, strain MK-256, produced histidase constitutively but retained the CnS character of its parent. This is illustrated in Fig. 2 where histidase production is measured in glucose containing media with glutamine as source of nitrogen.
The rate of histidase production is not significantly different in media containing excess glutamine, limiting glutamine as sole nitrogen source, or in a medium containing both glutamine and ammonia.
It has been shown in the preceding paper that in a Hut-constitutive strain with normal suceptibility to catabolite repression, Cn+, growth on excess or limiting glutamine in the absence of added ammonia leads to derepression o histidase (2). The results with MK-256 also demonstrate that ammonia accumulates in the medium even when glutamine is limited; in the Cn+ strain no accumulation of ammonia occurs under these growth conditions. CnR Revertants of Cns ikfutant-Some of the spontaneous revertants of the CnS strain MK-189 selected for their ability to grow on glucose-histidine had not recovered the full range of ability of the wild type to use amino acids as sources of nitrogen. In addition, these revertants (strains MK-204 and MK-208, Table IV) appeared to have the CnR character when subjected to the "tryptophan" spot test (2) in which CnR strains produce a yellow color on glucose-ammonia-tryptophan medium while Cn+ strains do not. We compared the sensitivity of these revertants to catabolite repression with that of their parent MK-189 (Table   v).
It can be seen that in all three strains ,Bgalactosidase responds normally to repression by glucose; histidase on the other hand in strains MK-204 and MK-208 is insensitive to repression by glucose even in media containing ammonia.
Thus, the control of histidase in strains MK-204 and MK-208 differs from that in their CnS parent (repressed in glucose-ammonia-histidine  (Table IV, compare strains MK-189 and MK-202); also some spontaneous revertants of RIK-189, selected for the ability to grow on the glucose-histidine medium, grew on all the media tested (Table IV, strain 203). Thus, the inability of MK-189 to grow on glucose plus any of these amino acids is probably due to a single mutation.
However, other revertants had only recovered the ability to use a more limited group of amino acids (MK-204, MK-208).
These will be discussed in the next section.
In order to substantiate the CnS character of strain MK-189 and to eliminate the need of adding histidine to the growth media, medium and in glucose-histidine medium) and from that in a Cn+ wild strain such as MK-53 (repressed in glucose-ammoniahistidine, but not in glucose-histidine).
Strains MK-204 and MK-208 have the CnR phenotype associated with some revertants of a glutamine-requiring mutant (2). We had previously shown that the genetic site responsible for the CnR character (gZnC) of the revertant of the glutamine requirer is closely linked to a site where mutations lead to the loss of glutamine synthetase (g2nA). We examined whether the mutation responsible for the CnR phenotype of strain MK-204 is due to a mutation at this site by using phage grown on MK-204 cells to transduce strain MK-604, a lysogenic derivative of the glutamine requiring strain MK-104 Apparently, a mutation in the glnC site is responsible for the CnR character of the revertants of the glutamine independent Cns strain described here and of the glutamine requiring CnS strain described in the previous paper. This finding is particularly interesting considering that entirely different procedures were used to obtain these mutants.
The former revertants were selected for their ability to use histidine as a nitrogen source in the presence of glucose, while the latter were selected for their ability to grow in the absence of glutamine.

Characteristics and Enzymatic
Composition of Cns and CnR Strains-As already noted, the Hut-constitutive derivative of the CnS strain MK-189 accumulates ammonia in the medium during growth on glutamine as sole source of nitrogen (see Fig. 2). This behavior suggested that the cells experience difficulty in the utilization of ammonia present at low concentration. Indeed, the CnS strain MK-189 was found to be incapable of growth in media containing ammonia at concentrations lower than 1 rnM as sole nitrogen source, whereas the prototroph can grow in media containing 0.01 mnl ammonia as sole nitrogen source.
Recent work by Meers et al. (4) has implicated two enzymes as necessary for synthesis of glutamate in K. aerogenes growing at low ammonia concentrations: glutamine synthctase and glutamine (amide) a+ketoglutarate amidotransferase oxido-reductase (NADP) (glutamate synthetase). Enzymatic analysis of extracts prepared from cells grown under a variety of conditions revealed that the glutamate synthetase activity is lacking in mutant MK-189 (Table VI, Experiments  6 and 7). Mixing experiments with extracts from MK-247, a prototroph, and MK-189 did not cause any decrease in MK-247 activity indicating that the absence of activity in the RIK-189 extract is not due to an inhibitor.
Sagatani et al. (9) isolated a mutant of Klebsiella unable to fis elementary nitrogen due to an inability to synthesize the glutamate synthetasc.
When Iye compared this mutant with its parent M5h1, we found that the mutant had also lost the ability to utilize histidine as a sole nitrogen source in the presence of glucose as well as its ability to grow on glucosamine medium, where nitrogen assimilation is limited by the slow hydrolysis of glucosamine.
Nagatani et al. refer to the genetic lesion in their mutant as asm-I.
Since MK-189 apparently has the same characteristics as asv-1, we refer to the mutation in ?\IK-189, which resulted in its inability to grow on limiting concentrations of ammonia, as asm-LOO.
Esamination of the CnR revertants of h/TK-189 (asm-200) revealed that these strains, MK-204 and MK-208, had lost the The cells were grown in media containing 0.2% of glucose as source of carbon and 0.2% of the indicated sources of nitrogen. Where indicated the nitrogen compound was added to the culture at a growth-limiting rate. The cells were disrupted and the extracts assayed for the three enzymatic activities.
Enzyme units are nanomoles of product formed per min. Gin-S, glutamine synthetase; Glut-S, glutamate synthetase; Glut-D, glutamate dehydrogenase. ability to grow in media containing ammonia as sole nitrogen ability to produce glutamine synthctasc is regulated (compare source, even when it was present at high concentration (33 nlM) Experiments 13 and 14). It produces this enzyme at a high level (Table IV).
The cells will grow in media supplemented with even in the presence of excess ammonia; it resembles in this L-glutamate, or with amino acids such as histidine, arginine, respect the donor strain 204 (conmarc Experiments 9 and 14). proline, or tryptophan which can provide glutamate either by It also resembles the donor, and differs from MK-104, by its their degradation or transamination reactions.
Thus, it appears deficiency of glutamate dehydrogenase (compare Experiments 1, that the CnR strains NK-204 and XK-208 have lost the ability 9, 13, and 14). It resembles the i,ecipient and differs from the to use ammonia for the synthesis of glutamate.
donor by its ability to produce glutamate synthetasc (compare The examination of cell extracts of MK-204 and MK-208 Experiments 9, 13, and 14). In summary, the transductant revealed the cause of the glutamate requirement (Table VI). selected for glutamine indcl)cndencc received from the donor t,he These strains, like their parent MKl89, still lack glutamate ability to produce high levels of glutamine synthctase even in the synthetase activity (Experiments 6, 7, 9 to la), and in addition, presence of excess ammonia, the CnR character, and the inability lack glutamate dehydrogenase activity.
Presumably, it is the to produce glutamate dehydrogenasc. We assume therefore that presence of glutamate dehydrogenasc in strain MK-189 (Experi-these characteristics may all be attributed to a single genetic ments 6 and 7) that enables this organism to grow in media con-trait, gZnC45, present in strain ilIK-204 that is linked to the site taining KHJ at concentrations greater than 1 mM without responsible for the inability to product glutamine synthetase in glutamate supplementation.
On the other hand, the transductant has redrogenase in strains MK-204 and MK-208 is apparently not due tained the abi1it.y of the recipient, absent in the donor, to ljroduce to the addition of glutamate to the medium since cells grown in a glutamate synthetase; this trait (nsm-SN) therefore does not medium to which glutamate is added slowly to limit the growth appear to be linked to the glnd site nor is its absence nccessarv for rate (Experiments 9 to 12) also lack this enzyme activity.  Table VI reveal interesting  This correlation between high glutamine synthetase and low  relationships between glutamine synthetase, glut'amate synthe-glutamate dehydrogenase levels is not limited to the strains detase, and glutamate dehydrogenase.
rived from the asm-$00 mutant since another strain with the CnR We have previously confirmed the results of other investiga-phenotype, selected as the revert,ant of glutamiile requiring tors that cells whose growth rate is limited by the availability of a mutant strain MK-93 (2), also contains a high level of glutamine nitrogen source contain more glutamine synthetase than those synthetase, but no glutamate dehgdrogenase n-hen grown on growing with the nitrogen source in excess (2). This is illus-excess ammonia (Experiment 16). Therefore, it appears that the trated by Experiments 1 to 3, in which cells with normal gluta-lack of glutamate dehydrogenase is generally associated with a mine synthetase were used. We can see that the level of this high level of glutamine synt'hetase (Experiments 2, 3, 9 to 12, 14, enzyme is higher in cells growing on limiting ammonia or limit-16, and 17). Conversely, a low level of glutamine sgnthctase is ing glutamine as the sole source of nitrogen than in cells growing associated with a high level of glutamate dehydrogenase. Thus, on excess ammonia.
The level of glutamate synthetase is not in strains with normal regulation of glutamine synthetase formagreatly altered, but the level of glutamate dehydrogenase is tion the level of glutamate dehydrogenase is high only when the greatly reduced by growth in the nit,rogen-deficient medium. cells are grown with excess ammonia (Experiments 1, 4 to 7). This confirms an earlier report by Meers et al. (4).
In strains deficient in glutamine synthctasr, the level of gluta-Experiments 4 and 5, in which a glutamate-requiring mutant mate dehydrogenase is high, even when the cells arc grown rrith was used, show that glutamate does not affect the production of the nitrogen source supplied at a growth-limiting rate (Experithese enzymes; their levels are approximately those found in the ments 13 and 15). wild type growing m-ith excess ammonia whether glutamate was The correlation of the reduced level of glutamate dchydroprovided in excess or as growth-limiting nutrilite. genase with the elevated glutamine synthetasc raises the question A deficiency in glutamate synthetase appears to prevent the whether a deficiency in glutamate dehydrogenase is responsible large increase in the level of glutaminc synthet,ase which occurs when glutamine is supplied as the growth rate-limiting nitrogen source (compare Experiments 3 and 8). Of particular interest are the effects of mutations in MK-204 and MK-208 which endow these revertants of the glutamate synthetase-deficient mutant with the ability to grow on glucosehistidine.
These strains, which are CnR, contain a high level of glutamine synthetase, even when grown in media containing excess ammonia (Experiments 9 t,o 12). They, like their parent MK-189, are deficient in glutamate synthetase, but in contrast for excessive production of the glutamine synthctasc.
To answer this question, we isolated from the glutamate synthetase-less mutant strain MK-189, mutants requiring glutamate, as described under "Experimental Procedure." One of thcsc mutants, MK-261, lacks both glutamate synthetase and glutamate dehydrogenase (Table VI, Experiment 18). We find that strain MK-261 has the CnS character of its parent MK-189, and produces glutamine synthetase at the usual low level in a medium containing excess ammonia. Some of the transductants obtained by treatment of cells of this strain with phage grown on to their parent they also lack glutamate dehydrogenase. wild type and selected for the ability to grow on a medium We have shown in the previous section that phage grown on without glutamate had recovered only the glutamate synthetase the CnR strain MK-204 transduce the CnR character to the gluta-(strain MK-270, Experiment 19). Strain AIR-270 has the Cn+ mine-requiring CnS strain MK-104 together with glut'amine character of the wild tyllc, and also resembles the wild tyl'c in its independence.
WC find now that the transductant strain low level of glutaminc synthetase on excess ammonia (Esperi-MK-257 differs from the recipient by the manner in which it,s ment 19). It appears therefore that a deficiency in glutamate