INTRODUCTION

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Virtually all the nitrogenous compounds in an enteric bacterium derive their nitrogen atoms from either glutamate or glutamine. It has been estimated that about 88% of the cellular nitrogen in Escherichia coli is derived from glutamate and the remaining 12% is derived from glutamine (40). Thus, glutamine and, especially, glutamate are the key intermediates in cellular nitrogen metabolism. However, neither glutamate nor glutamine is capable of supporting growth at a rate comparable to that supported by ammonium salts when provided as the sole nitrogen source, probably because of inefficient transport of both compounds. Clearly, ammonium is the preferred nitrogen source for the enteric bacteria. In the presence of ammonium, there is a strong repression of many systems that allow the cells to use alternative nitrogen sources, such as amino acids, inorganic compounds, and urea (29).

Ammonia can be fixed onto the carbon skeleton of -ketoglutarate to form glutamate by two different pathways in the enteric bacteria. It can be fixed directly onto -ketoglutarate in an NADPH-dependent reaction by glutamate dehydrogenase (GDH). It can also be fixed indirectly by first being attached to glutamate to form glutamine by an ATP-dependent glutamine synthetase (GS), followed by the transfer of the amide nitrogen onto -ketoglutarate by an NADPH-dependent glutamate synthase, also called glutamate-oxoglutarate amidotransferase (GOGAT). No other pathway allows significant net assimilation of ammonia into glutamate. Mutants lacking both GDH and GOGAT are glutamate auxotrophs, requiring glutamate, a compound that can be degraded to glutamate, or a compound that can transaminate its nitrogen to -ketoglutarate (9).

Under conditions of ammonia-limited growth, GDH plays a relatively minor role in nitrogen assimilation because of its high K_m for ammonium (33). In Klebsiella spp., this role is further minimized because GDH is strongly repressed under conditions of nitrogen limitation (33). In fact, mutants lacking GDH have no altered growth phenotype in glucose minimal medium and show a competitive disadvantage relative to the wild type only during energy limitation (21). In other words, GOGAT is able to provide the cell's total glutamate supply, and during ammonia-limited growth, the indirect pathway (via glutamine and GOGAT) becomes essential (9). Under conditions of ammonia excess, either pathway is sufficient for glutamate synthesis.

The response to nitrogen excess or limitation is determined by the intracellular concentration of glutamine (23). Glutamate seems to play no role in signaling nitrogen excess or limitation. However, glutamate does play an important role in the osmoregulation of the cell, especially as the counterion that allows the accumulation of intracellular potassium (42). Glutamate and glutamine play very different regulatory roles in the cell despite their proximity in metabolic pathways. Thus, it is important that the cell have mechanisms to adjust the pools of glutamate and glutamine independent of each other and to do so during changes in nitrogen availability or osmotic challenge. The glutamate pool is filled by GDH and GOGAT and depleted by GS and general biosynthetic reactions. The glutamine pool is filled by GS and depleted largely by GOGAT and some biosynthetic reactions. There are several glutaminases reported in enteric bacteria, but it has been suggested that these are relatively inefficient in comparison to GOGAT (40). The key enzymes for maintaining the proper relative sizes of the pools of glutamate and glutamine are GS (which depletes glutamate and produces glutamine) and GOGAT (which depletes glutamine and produces glutamate).

Mutants lacking GS activity cannot accumulate glutamine (the signal of nitrogen excess) except by transport, and their nitrogen regulation is blind to exogenous ammonia (6). Such mutants mount a nitrogen starvation response (derepress the Ntr system) even in the presence of high concentrations of ammonia. This understanding was initially confused by the fact that polar mutations in glnA (the structural gene for GS) fail to express the ntrC gene product and thus mount a nitrogen excess response (no Ntr system expression) even in the absence of ammonia.

GOGAT is a heterodimeric protein whose subunits are encoded by the gltB and gltD genes. The gltB gene codes for the larger glutaminase subunit, and gltD codes for the smaller transaminase subunit analogous to GDH. Mutants that lack GOGAT cannot deplete glutamine except by biosynthetic reactions. Moreover, when ammonia is limiting (too dilute for efficient fixation by GDH), mutants that lack GOGAT cannot accumulate glutamate except by transport, transamination, the residual activity of GDH, or the weak glutaminases of the cell. Such mutants fail to grow on low concentrations of ammonia, grow poorly on glutamine, and grow poorly or not at all on a variety of nitrogen-containing compounds that can be catabolized to ammonia, glutamate, or both (9). When cells with a gltB or gltD mutation (lacking GOGAT activity) are grown with histidine as the sole nitrogen source, growth is very slow, suggesting a defect in activating the histidine utilization (hut) operons. The original explanation for this failure to grow with histidine as the sole nitrogen source was based on arguments about the size of the pool of glutamine. In the absence of GOGAT activity, the size of the glutamine pool would increase and could not be depleted, thus preventing activation of the Ntr system and consequently of histidase formation (2, 40). However, this leads to the paradoxical predictions of glutamine pools that are too high to allow derepression of the Ntr system but too low to allow growth. The discovery of a third gene downstream in the gltBD operon, gltF, led to an alternative explanation in which the GltF product was essential for nitrogen regulation and gltBD mutations might be polar on gltF expression (10, 11).

The experiments described here were designed to examine the growth defects of gltBD mutations and to determine the effects of the gltF gene on nitrogen regulation in the enteric bacteria.

	MATERIALS AND METHODS

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Bacterial strains. Descriptions and genotypes of the bacterial strains used are listed in Table 1. The species Klebsiella aerogenes has been subsumed into the species Klebsiella pneumoniae; however, we have retained the older name for strains derived from strain W70 for historical reasons and to distinguish it from the nitrogen-fixing K. pneumoniae strain M5aL (which is properly Klebsiella oxytoca), from which it differs considerably. All K. aerogenes strains are derived from strain W70, all K. pneumoniae strains are derived from strain M5aL, all E. coli strains are derived from strain K-12, and all Salmonella enterica serovar Typhimurium strains are derived from strain LT-2.

Growth media. Bacterial cultures were grown either in rich (LB) medium or in W4 minimal medium (W salts [4] adjusted to an initial pH of 7.4). Minimal medium was supplemented with 0.4% (wt/vol) glucose as a carbon source and one or more of the following nitrogen sources: ammonium sulfate (0.2%); L-glutamine (0.2, 0.1, or 0.04%); L-serine, L-arginine, or potassium nitrate (each at 0.2%); or the monosodium salt of L-glutamate (0.2 or 0.4%). Thiamine HCl (10 µg/ml) was used to supplement thiamine auxotrophs where necessary. Media for plasmid and transposon selection and maintenance were supplemented with antibiotics as follows: ampicillin (75 µg/ml for E. coli strains and K. aerogenes strains carrying the [bla]-2 deletion and 1,000 µg/ml for bla⁺ strains of K. aerogenes), spectinomycin (100 µg/ml), streptomycin (50 µg/ml), kanamycin sulfate (50 µg/ml), and tetracycline (30 µg/ml). Media for selection of strains bearing a single copy of the  cartridge (38) on the chromosome were supplemented with spectinomycin and streptomycin at 100 and 50 µg/ml, respectively, for some strains (W3110, YMC10, and EB4504) or at 50 and 25 µg/ml, respectively, for others (EB566, EB4566, and EB4722). Whenever glutamine was used as the sole nitrogen source, it was Calbiochem A grade.

Genetic and molecular techniques. The hutUp-lacZ fusion was transferred from the plasmid pCB540 to the phage RZ-5 and then to the chromosome of W3110 essentially as described by Grove and Gunsalus (20). Briefly, RZ-5 was grown on a YMC10 transformant carrying pCB540, and the lysate thus obtained was used as a source of the double-crossover recombinant phages by transducing an attB⁺ E. coli strain to Ap^r and screening for stable lysogens. Stable lysogens were defined as those which retained Ap^r at a frequency of 100% after three rounds of subculture on LB medium in the absence of ampicillin. Recombinant phages spontaneously released from the stable lysogens were used to prepare a lysate of pure RZ-5CB540, which was used to transduce W3110 to Ap^r.

Cloning and sequencing of the gltBD region from K. aerogenes The wild-type gltB gene and surrounding region was cloned from K. aerogenes strain KC1467 using the in vivo Mu lysate technique (19). Strain KB2560 (a K. aerogenes strain that lacks both GOGAT and GDH and is therefore auxotrophic for glutamate) was transduced with the lysate from KC1467 with selection for glutamate prototrophs (Glt⁺). Glt⁺ transductants were screened for growth on glucose minimal medium supplemented with serine to 0.2%, a growth phenotype which distinguishes between Gogat⁺ strains of K. aerogenes, which grow, and Gogat strains, which do not (9). One of the recovered plasmids that was found to confer the Gogat⁺ phenotype on KB2560 (pCB507) carried approximately 20 kb of inserted DNA. pCB507 was digested with EcoRI, from which a 12-kb fragment was recovered and cloned into the EcoRI site of either pUC19 or pGB2, yielding pCB536 and its oppositely oriented subclone pCB537 (in pUC19) or pCB548 (in pGB2). Although the high-copy-number plasmids pCB536 and pCB537 could not be stably maintained in K. aerogenes, the low-copy-number plasmid pCB548 was maintained stably in K. aerogenes and was found to complement the glutamate auxotrophy of KB630. Subclones derived from pCB536 and pCB537 and maintained in E. coli (pCB593, pCB620, and pCB633) were used as templates for automated sequencing of both strands of the DNA, from which the sequence of 7,930 bp was determined.

Complementation and marker rescue analysis. The ability of plasmids carrying portions of the wild-type K. aerogenes gltBD operon to complement the glutamate auxotrophy of KB630 (gltB200 gdhA1) or to recombine with gltB200 to yield gltB⁺ recombinants was tested as follows. Strain KB630 was transformed to drug resistance with the test plasmid. Drug-resistant transformants were selected on LB agar supplemented with either a mixture of streptomycin and spectinomycin (when pCB548 was used) or ampicillin (when pUC-based plasmids were used.) Fifty or more isolated colonies were then patched onto glucose ammonia minimal medium (without glutamate) with sterile toothpicks. Complementation was indicated if all patches contained solid and uniform growth after 16 to 48 h of incubation. Recombination was indicated if 30% or more of the patches contained one or more isolated colonies. If none of the patches showed uniform growth or isolated colonies, the plasmid was considered to be incapable of complementation or recombination.

Southern blot analysis. Southern blotting and hybridization were carried out as described in the Genius nonradioactive labeling and detection kit (Boehringer Mannheim) using digoxigenin-labeled probes obtained with the random priming method. Low-stringency hybridization was carried out at 42°C with washes in 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at room temperature. Moderate-stringency hybridization was carried out at 58°C with washes in 2× SSC and 0.5× SSC at 58°C. High-stringency hybridization was carried out at 65°C with washes in 2× SSC and 0.5× SSC at 65°C. The probe used to detect homologues of the gltF gene was an 830-bp PCR product corresponding to the E. coli gltF gene (as indicated in the figures). The probe used to detect homologues of the gltD gene was an 882-bp PCR product obtained by amplification of the K. aerogenes gltD gene (as indicated in the figures). Nylon filters were stripped for reblotting using the alkaline stripping method suggested by the kit's manufacturer.

Mutagenesis of E. coli gltF. The gltF1:: mutation was constructed in vitro and introduced into the E. coli chromosome by reverse genetics essentially as described by Horton and Pease (22). The result of this process was a fragment containing all of the DNA sequences surrounding gltF but with the coding sequence of gltF replaced by a BamHI site. This fragment was cloned into pBS SK(+), resulting in pCB1055, which carried the gltF1 allele. The  fragment, specifying resistance to streptomycin and spectinomycin, was purified from BamHI-digested pPH45 (38) and ligated into the BamHI site in pCB1055, yielding pCB1063 carrying gltF1::.

Enzyme assays. Cultures of K. aerogenes and E. coli were grown at 30 and 37°C, respectively, in minimal medium supplemented as indicated in the tables to a density of 50 Klett units (filter 54) except where indicated. Cells were collected by centrifugation, washed once with 1% (wt/vol) KCl, and resuspended at 10-fold concentration (about 1 mg of protein/ml) in 1% KCl. The histidase, GOGAT, and -galactosidase assays have been described previously (28). Specific activities are reported as nanomoles of product formed (or substrate degraded) per minute per milligram of protein.

Nucleotide sequence accession number. The DNA sequence of the gltBD region from K. aerogenes was determined using the automated fluorescent dye termination method at the University of Michigan Core facility and has been deposited in GenBank under accession no. AY035435.

	RESULTS

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The gltBD operon of K. aerogenes. The gltBD region from K. aerogenes was cloned as described in Materials and Methods, and the DNA sequence of 7,930 bp from the region was determined. The key features of this sequence are summarized in Fig. 1. There are two open reading frames (ORFs) which are highly similar to the gltB and gltD ORFs of E. coli (95 and 93% identity over 1,486 and 473 amino acid residues, respectively), except for a patch of 8 residues (amino acids 584 to 591) in GltB. The K. aerogenes gltB and gltD ORFs are separated by a 12-bp spacer, slightly smaller than the 15-bp spacer found between the E. coli gltB and gltD genes. An oppositely directed ORF of 324 amino acids (orfB) lies 60 bp downstream from the GltD ORF. The deduced amino acid sequence of orfB is 74% identical (but five residues longer) than the yhcJ ORF in E. coli. In E. coli, yhcJ lies more than 9 kb downstream from gltD (7).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Comparison of the gltBD regions from E. coli and K. aerogenes. Homologous ORFs are indicated by similar hatching. The solid lines in the lower portion represent the DNA sequences present in the plasmids listed. E, Sm, S, P, Bs, and K represent restriction enzyme cleavage sites for EcoRI, SmaI, SalI, PstI, BssHII, and KpnI, respectively. (Note that additional sites exist in the cloned DNA for many of the indicated restriction enzymes.) Ability (+) or inability () to form recombinants by marker rescue with the gltB200 allele of K. aerogenes is indicated.

Characterization of the gltB200 mutation. The gltB200 mutation (formerly asm-200) was originally isolated as a chemically induced mutant that was unable to use urocanate as the sole nitrogen source (9). Further studies showed that the phenotype of gltB200 was similar to those of other gltB mutations. Strains carrying gltB200 grew poorly with histidine as the sole nitrogen source, failed to grow with serine as the sole nitrogen source, lacked GOGAT activity, and caused an auxotrophic requirement for glutamate in strains carrying the gdhA1 mutation. (The gdhA1 mutation eliminates GDH activity, the only enzyme other than GOGAT capable of net glutamate synthesis from ammonia [9].) Cloned fragments containing portions of the gltBD operon were tested for the ability to give Glt⁺ recombinants with the gltB200 mutation. The data presented in Fig. 1 can be summarized by saying that all clones that contained the segment of gltB DNA from 2,689 to 2,881 bp downstream from the start of the gltB ORF gave rise to Glt⁺ recombinants. This corresponds to the BssHI and KpnI sites at positions 6.45 and 6.64 in Fig. 1. All clones tested that lacked this segment failed to yield recombinants with the gltB200 mutation. As expected, the frequency of such "marker rescue" was found to be lower for clones carrying short regions of homology than for those with more extensive regions of homology, and the introduction of a recA3011 mutation reduced the frequency of Glt⁺ recombinants. Thus, it appeared that the mutation responsible for the Glt phenotype (gltB200) must lie within the region from 2,689 to 2,881 bp downstream from the start of the gltB ORF.

Characterization of the gltD702 mutation. The glt-702 mutation was originally isolated as a Tn5 insertion mutant of strain KC1419 (3) that was unable to use urea as the sole nitrogen source. We have shown that the phenotype of glt-702::Tn5 is similar to that of gltB200 in that strains carrying glt-702::Tn5 grew poorly with urea or histidine as the sole nitrogen source, failed to grow with serine as the sole nitrogen source, lacked GOGAT activity, and caused an auxotrophic requirement for glutamate in strains carrying the gdhA1 mutation (data not shown). Mapping studies showed that the glt-702::Tn5 mutation was about 20% linked to argG2 by P1-mediated generalized transduction, the same as the linkage of gltB200 and argG2 (16). Thus, we assumed that glt-702 was a mutation in the gltBD operon.

Absence of gltF in K. aerogenes. Since no gene homologous to gltF was found in the region immediately downstream of the K. aerogenes gltD gene, Southern analysis was used to see if a homologue of gltF existed anywhere in the genomes of K. aerogenes and other selected enteric bacteria. DNA from strains of K. aerogenes, K. pneumoniae (K. oxytoca), and S. enterica serovar Typhimurium was hybridized with a probe corresponding to the E. coli gltF ORF. DNA from E. coli (from which the gltF probe was derived) served as a positive control. When carried out under conditions of low hybridization stringency, a band of the size expected for E. coli gltF was observed in the E. coli lane (Fig. 2A). Less intense, smeared signals were also observed at this low stringency, which likely correspond to weaker interactions of the gltF probe with E. coli DNA other than gltF. No other DNA contained material which strongly hybridized to the gltF probe; however, as with the E. coli samples, less intense, smeared signals were obtained with the other samples. This was especially true for the S. enterica serovar Typhimurium sample and leaves open the question of whether a gene with some homology to gltF might exist somewhere in the S. enterica serovar Typhimurium genome.

View larger version (49K): [in this window] [in a new window]   FIG. 2.   Southern blot of chromosomal DNA digested with either EcoRI or PstI. (A) The probe was derived from an 830-bp fragment corresponding to the E. coli gltF ORF as depicted at the top. Hybridization conditions were low stringency (see Materials and Methods). (B) The filter was stripped and reprobed with a probe that was derived from an 882-bp fragment corresponding to the 3' end of the K. aerogenes gltD ORF as depicted at the top. Hybridization conditions were moderate stringency. Lanes 1 through 9 contained chromosomal DNA from E. coli strain W3110 (E.c.), S. enterica serovar Typhimurium strain LT-2 (S.t.), K. aerogenes strain MK1 (K.a.), and K. pneumoniae strain KP4387 (K.p.), which is derived from strain M5aL. Lane M contained gltF standards of 3.8 and 0.89 kb and a gltD standard of 6.4 kb.

The role of gltF in E. coli Since homologues of gltF appear to be absent from the chromosomes of K. aerogenes and K. pneumoniae, we questioned whether the gltF gene plays a species-specific role in the function of the Ntr system in E. coli. E. coli strain EB4613 carries the psiQ39 mutation (34), in which the Mu d1-1734 phage is inserted into the gltD gene approximately one-third of the gene's length from its carboxy (3') terminus. (Hereafter, we will refer to this mutation as gltD39::Mu d1-1734 or simply gltD39.) As expected of a gltD strain, EB4613 lacks GOGAT activity (Gogat phenotype) and fails to grow with arginine as the sole nitrogen source (Asm phenotype). EB4613 was transformed with pCB548, a low-copy-number plasmid carrying the gltBD operon from K. aerogenes. The resulting strain, EB4615, regained GOGAT activity and was able to use arginine as the sole nitrogen source. Thus, the presence of gltBD from K. aerogenes seemed sufficient to restore both Gogat⁺ and Asm⁺ phenotypes, even in the absence of an added gltF gene. If gltF is indeed transcribed from a promoter located upstream of the polar gltD39 mutation, then restoration of the Asm⁺ phenotype by a plasmid that carried only gltB and gltD (and not gltF) suggests that there is no clear role for gltF in the derepression of the Ntr system.

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Southern blot analysis of EcoRI-digested chromosomal DNA from wild-type E. coli strains and derivatives carrying the gltF1:: mutation. (A) The probe was derived from an 830-bp fragment corresponding to the gltF ORF as depicted at the top. Hybridization conditions were high stringency. (B) The filter was stripped and reprobed with a probe that was derived from an 882-bp fragment corresponding to the 3' end of the K. aerogenes gltD ORF. Hybridization conditions were moderate stringency. Lanes 2, 3, and 5 contained DNA from three different E. coli "wild types," EB4566 (derived from W3110), YMC10, and EB566. Lanes 1, 4, and 6 contained DNA from the gltF1:: mutants derived from these three strains (EB4582, EB4534, and EB4539, respectively).

View this table: [in this window] [in a new window]   TABLE 2.   Effect of gltF1 on enzyme levels and growth rates in four E. coli backgrounds

GOGAT mutants have difficulty deriving glutamate from glutamine. It has long been assumed that GOGAT is the principal route by which glutamine is degraded in both K. aerogenes (2) and E. coli (40). However few data have been presented to document this assumption. When wild-type E. coli strain W3110 was inoculated into glucose minimal medium with glutamine (0.1% wt/vol) as the sole nitrogen source (Ggln), it grew rapidly to high density, with a doubling time of 75 min (Fig. 4A). When the gltD mutant strain BE3471 (otherwise isogenic with W3110) was inoculated into Ggln, the growth was biphasic (Fig. 4B), with initial rapid growth (doubling time, about 85 min) followed by slow but still exponential growth (doubling time, about 658 min). Increasing the initial concentration of glutamine from 0.1 to 0.2% increased both the initial growth rate and the cell density at which the shift from fast to slow growth occurred. Conversely, lowering the glutamine from 0.1 to 0.04% decreased both the initial growth rate and the cell density at which the shift from fast to slow growth occurred (data not shown). The growth rate of the second, slow phase of growth was independent of the glutamine concentration. Such biphasic growth kinetics are reminiscent of diauxic growth, where more rapidly utilized nutrients are depleted from the growth medium before the utilization of growth rate-limiting nutrients. When a culture of strain BE3471 (gltD39) was supplemented with additional glucose or additional glutamine, the added glutamine extended the period of rapid growth whereas the glucose had no effect (data not shown). Thus, the growth rate limitation imposed on the GOGAT-deficient E. coli strain growing in Ggln was eased by the presence of additional glutamine.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Growth of E. coli strains W3110 (gltD+) (A) and BE3471 (gltD39) (B) in glucose minimal medium at 37°C with the nitrogen sources and supplements indicated. G, glucose at 0.4% (wt/vol); gln, glutamine at 0.1% (wt/vol); glt, glutamate at 0.2% (wt/vol); N1, 1 mM ammonia (added as 0.5 mM ammonium sulfate).

Growth of K. aerogenes GOGAT mutants. When wild-type K. aerogenes strain KC1043 was grown in Ggln medium, it grew rapidly (73-min doubling time) to high density (Fig. 5). When KC2561, a gltB derivative of KC1043, was grown in Ggln, growth was much slower (215-min doubling time) but monophasic (Fig. 5). As was true for the E. coli GOGAT-deficient strains, growth of the K. aerogenes gltB mutant KC2561 was faster when the Ggln medium was supplemented with either glutamate (not shown) or 1 mM ammonia (Fig. 5). The effectiveness of 1 mM ammonia was surprising, since GDH is strongly repressed in K. aerogenes by the Ntr system acting via NAC (Table 3). However, it is clear that GDH is required for the ammonia-mediated effect. When the GOGAT-deficient strain KC2561 was compared with strain KC3182, which lacks both GOGAT and GDH, ammonia did not increase the growth rate above that seen in Ggln medium (Fig. 5). A strain lacking GDH only (GOGAT was present) grew like the wild type in all the media tested here (data not shown). Thus, for K. aerogenes as for E. coli, growth of a GOGAT-deficient mutant on glutamine as the sole nitrogen source results in a growth rate limitation for glutamate. This argument is supported by an earlier observation (2) that the growth rate of a GOGAT-deficient mutant on Ggln medium is much lower (3- to 4-h doubling time) than that of a GOGAT-deficient mutant that also lacks GS, a glutamate-consuming enzyme (98-min doubling time).

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Growth of K. aerogenes strains. Squares, strain KC1043 (wild type); circles, KC2561 (gltB200); triangles, KC3182 (gltB200 gdhA2). Growth was in minimal medium with 0.4% (wt/vol) glucose as the carbon source and 0.1% glutamine as a nitrogen source. Open symbols, no other additions; solid symbols, 1 mM ammonia was also present (as 0.5 mM ammonium sulfate).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Inadequacy of the GltF hypothesis. Since the report of the original K. pneumoniae asm-1 mutation in 1971 (36), the cause of the inability of enteric bacteria lacking GOGAT activity (Gogat) to assimilate the nitrogen from several alternative nitrogen sources (Asm phenotype) has been open to speculation. One aspect of the problem was clear from the outset: Gogat mutants are unable to assimilate ammonium when it is present in the medium (or generated by catabolism) at levels too low to be used by GDH, whose K_m for ammonium is quite high. Thus, the growth of Gogat mutants on low ammonia is limited by glutamate availability. But there is also one report that Gogat mutants were unable to derepress the Ntr system and hence the Ntr-dependent operons that code for the catabolism of a variety of alternative nitrogen sources, such as histidine (9). It has been argued that GOGAT activity is required to reduce the size of the intracellular glutamine pool so that Ntr derepression can occur (2). Although a model based on glutamine pools superficially explains the Asm and Ntr phenotypes of Gogat strains, this model leaves us with a paradox. GOGAT-deficient cells must have a glutamine pool that is too high to allow activation of the Ntr system but too low to allow growth. This difficulty is compounded by the observation that Gogat mutants of K. aerogenes grow with glutamine as the sole exogenously supplied nitrogen source but apparently cannot grow on the endogenous pool of glutamine which accumulates in the presence of alternative nitrogen sources.

The glutamate starvation hypothesis. If the GltF model must be discarded, that leaves the glutamine imbalance model with its glutamine paradox to explain the Ntr phenotype of Gogat mutants. Our data provide a clarification of this apparent paradox by suggesting that Gogat mutants fail to grow on alternative nitrogen sources because they are starved for glutamate. In other words, the inability to derepress the Ntr system of E. coli is not a cause of the phenotype but rather an effect of it. If growth is limited by the supply of glutamate, then any supply of glutamine will, by definition, be excess. Thus, Gogat mutants fail to grow on Ntr-dependent nitrogen sources for the same reason they fail to grow on low concentrations of ammonia: they are glutamate starved.

Open questions about glutamate synthesis. The data presented here deal directly with effects of aberrant regulation of glutamate metabolism, but they also raise a number of questions about the normal regulation of glutamate formation. It is clear that gltBD expression requires activation by the leucine-responsive regulatory protein Lrp (15). The data presented here show clearly that NAC is capable of severely repressing gltBD expression (Table 2). We and others have noted a strong repression of both gltBD and gdhA expression in cells grown in broth (8). For gltBD, this repression can be explained by the lack of Lrp under such conditions (15), but for gdhA (which codes for GDH), Lrp is not necessary (unpublished observations). The severe repression of gdhA expression by broth is independent of the severe NAC-mediated repression of gdhA, but the nature of the effector responsible for broth repression of gdhA is unknown. The data in Table 3 contain one further unexpected finding: when E. coli GOGAT mutants were starved for glutamate (by growing them with glutamine as the sole nitrogen source), GDH levels increased dramatically to as much as 10-fold that seen in cells grown in high ammonia. The mechanism of this increase is unknown, but the increase has important consequences for cells limited for glutamate. GOGAT mutants are dependent on GDH for the assimilation of ammonia into glutamate. At concentrations of ammonium lower than about 1 mM, K. aerogenes NAC causes severe repression of GDH, and the poor K_m of GDH for ammonia means that the residual GDH is ineffective. In E. coli and S. enterica serovar Typhimurium, GDH is repressed either only slightly or not at all by NAC, allowing GOGAT mutants to grow with ammonia concentrations as low as 100 µM. It has long been known that mutations (or high-copy-number clones) that increase the production of GDH allow growth at even lower concentrations of ammonia (5). However, except for one class of mutation that creates a new, stronger promoter for gdhA in K. aerogenes (25), the nature of these GDH overexpression mutations is unknown. Given the central role of glutamate in biosynthesis and osmoregulation, it is not surprising that its synthesis is subject to multiple forms of regulation. What is surprising is how little is known about that regulation.

A reinterpretation of the data that led to the GltF hypothesis. Finally, the data presented here suggest an explanation for some of the irreproducible results that have been reported regarding regulatory effects that are or are not seen in cells grown with 0.2% (and perhaps 0.1%) glutamine as the sole nitrogen source. The effect is clearest in the E. coli gltD mutant growing with glutamine as the sole nitrogen source. When the cell density is sufficiently low, the spontaneous decomposition of the glutamine in the medium provides enough glutamate (and ammonia) to allow rapid growth. When the cell density reaches a critical value, dependent on the concentration of glutamine, the supply of glutamate and ammonia becomes insufficient and the cells are truly dependent on glutamine alone as the nitrogen source. If we extrapolate that finding to wild-type cells, we can assume that, at low cell densities, cells are growing on a mixture of glutamine and small amounts of glutamate and ammonia; at higher densities, the nitrogen source is glutamine alone. Thus, early in the growth of the culture, Ntr-activated enzymes (histidase) are not formed and Ntr-repressed enzymes (GDH) are formed and accumulate. At a cell density of about 10⁸ cells/ml, Ntr-activated enzymes (histidase) are formed rapidly and accumulate while Ntr-repressed enzymes (GDH) stop accumulating and begin to be diluted. Thus, if cells are harvested 1 or 2 generations after the transition occurs, they will still have substantial quantities of GDH and will have already accumulated substantial quantities of histidase. Moreover, the exact point at which the transition occurs will depend on the size of the inoculum, the growth rate of the cells, and the concentration and quality of the glutamine in the medium. In short, small changes in the growth conditions can have the result that 0.2% glutamine is no longer barely derepressing and may make 0.2% glutamine either a strongly derepressing or nonderepressing medium. This may explain why effects that actually result from slight variations in growth rates and conditions have been attributed to gltF.