Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae.

We have investigated whether simultaneous modification of cofactor metabolism and glycerol in a strain of Saccharomyces cerevisiae can eliminate glycerol synthesis during ethanol production. Two strains, S812 (gpd1Δ gpd2Δ PGK1p-GLT1) and LE17 (gpd1Δ gpd2Δ PGK1p-GLT1 PGKp-STL1) were generated that showed a 8 and 8.2 % increase in the ethanol yield, respectively, compared to the wild type KAM-2 strain. The ethanol titer was improved from 90.4 g/l for KAM-2 to 97.6 g/l for S812 and 97.8 g/l for LE17, respectively. These results provide a new insight into rationalization of metabolic engineering strategies for improvement of ethanol yield through elimination of glycerol production.

Over-expressing GLT1 in a gpd2Delta mutant of Saccharomyces cerevisiae to improve ethanol production.

We constructed two recombinant strains of Saccharomyces cerevisiae in which the GPD2 gene was deleted using a one-step gene replacement method to minimize formation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene by a two-step gene replacement method to overcome the redox-imbalancing problem in the genetically modified strains. The result of anaerobic batch fermentations showed that the rate of growth and glucose consumption of the KAM-5 (MATalpha ura3 gpd2Delta::RPT) strain were slower than the original strain, and the KAM-13 (MATalpha ura3 gpd2Delta::RPT P ( PGK ) -GLT1) strain, however, was indistinguishable compared to the original strain using the same criteria, as analyzed. On the other hand, when compared to the original strain, there were 32 and 38% reduction in glycerol formation for KAM-5 and KAM-13, respectively. Ethanol production increased by 8.6% for KAM-5 and 13.4% for KAM-13. Dramatic reduction in acetate and pyruvic acid was also observed in both mutants compared to the original strains. Although gene GPD2 is responsible for the glycerol synthesis, the mutant KAM-13, in which glycerol formation was substantially reduced, was able to cope and maintain osmoregulation and redox balance and have increased ethanol production under anaerobic fermentations. The result verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and over-expressing the GLT1 gene along with reconstituted nicotinamide adenine dinucleotide metabolism.

Reduced glial fibrillary acidic protein and glutamine synthetase expression in astrocytes and Bergmann glial cells in the rat cerebellum caused by delta(9)-tetrahydrocannabinol administration during development.

In this study we analyzed the responses of cerebellar astroglial cells to pre- and perinatal delta(9)-tetrahydrocannabinol (THC) exposure in three postnatal ages and both sexes. To determine whether THC during development directly modifies astroglial growth, this study investigated the effects of THC on astroglial morphological changes and on the expression of specific astroglial markers (glial fibrillary acidic protein: GFAP and glutamine synthetase: GS). Our results demonstrated that the administration of THC during development has deleterious effects on astroglial maturation in the cerebellum. These results also indicate that THC might interfere with astroglial differentiation in a way dependent on sex. The effect of cannabinoids on the development of cerebellar astroglial cells (astrocytes and Bergmann glial cells) is to reduce protein synthesis, since both GFAP and GS decreased in astroglial cells, not only during THC exposure but also in adult ages. Our data suggest that pre- and perinatal THC exposure directly interferes with astroglial maturation by disrupting normal cytoskeletal formation, as indicated by the irregular disposition of GFAP and the lower GFAP expression observed at all the ages studied. THC exposure during development may also modulate glutamatergic nervous activity since GS expression is reduced in THC-exposed brains. GS expression increased progressively after THC withdrawal, but GS expression had still not reached control values two months after THC withdrawal. This indicates that glutamate uptake is lower in glial cells exposed to THC, since GS expression is lower than in older controls. Consequently, glutamatergic neurotransmission may be affected by cannabinoid exposure during gestation. Therefore, cannabinoids exert developmental toxicity, at least on astroglial cells, which could contribute to fetal brain growth retardation.

Stress-response sigma factor sigma(H) directs expression of the gltB gene encoding glutamate synthase in Streptomyces coelicolor A3(2).

Using the previously established Escherichia coli two-plasmid system, we identified a promoter recognized by the Streptomyces coelicolor stress-response sigma factor sigma(H). The promoter directed expression of the gltB gene, encoding a protein with considerable homology with large subunit of glutamate synthases. S1-nuclease mapping using RNA prepared from S. coelicolor identified an identical transcription start point corresponding to the promoter. The level of the transcript from this promoter was substantially reduced in a S. coelicolor sigH mutant. In addition to this sigH-dependent gltBp2 promoter, expression of the S. coelicolor gltB gene was directed by two other promoters, gltBp1 and gltBp3, independent upon sigH. S. coelicolor core RNA polymerase, after complementation with sigma(H), was able to recognize the gltBp2 promoter in vitro. These results suggested that the S. coelicolor gltB gene is under the control of stress-response sigma(H).

Role of AP-1 in the coordinate induction of rat glutamate-cysteine ligase and glutathione synthetase by tert-butylhydroquinone.

GSH synthesis occurs via two enzymatic steps catalyzed by glutamate-cysteine ligase (GCL, made up of two subunits) and GSH synthetase (GS). Recently, we described coordinate induction of GCL subunits and GS. To study GS transcriptional regulation, we have cloned and characterized a 2.2-kb 5'-flanking region of the rat GS (GenBank accession number AF333982). One transcriptional start site is located at 51 nucleotides upstream of the translational start site. The rat GS promoter drove efficiently luciferase expression in H4IIE cells. Sequential deletion analysis revealed DNA regions that are involved in positive and negative regulation. One repressor identified was NF1. tert-Butylhydroquinone (TBH) exerted a dose- and time-dependent increase in the mRNA level and promoter activity of both GCL subunits and GS. TBH increased protein binding to several regions of the GS promoter, c-jun expression, and activator protein 1 (AP-1) binding activity to several of the putative AP-1-binding sites of the GS promoter. Blocking AP-1 binding with dominant-negative c-jun led to decreased basal expression and significantly blocked the TBH-induced increase in promoter activity and mRNA level of all three genes. In conclusion, AP-1 is required for basal expression of GCL and GS; while NF1 serves as a repressor of GS, increased AP-1 transactivation is the predominant mechanism for coordinate induction of GCL and GS expression by TBH.

Fine tuning of glutamate uptake and degradation in glial cells: common transcriptional regulation of GLAST1 and GS.

Glutamate is the major excitatory neurotransmitter in the mammalian brain and retina, and glutamate uptake is essential for the normal function of glutamatergic synapses in the retina. As summarized here, all neuronal and macroglial cells of the retina express high-affinity glutamate transporters. GLAST1 is expressed in glial cells, whereas GLT1 and EAAC1 are neuronal. Glutamate uptake studies in intact retina revealed that Müller glial cells dominate the total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. A prerequisite for an effective glutamate-glutamine cycle in glial cells would be the regulated coordination between glutamate uptake and glutamate degradation. Using cultured retinal Müller glial cells, we demonstrate that protein expression of both, GLAST1 and glutamine synthetase, are inducible by the glucocorticoid hormone cortisol. These results suggest a common transcriptional regulation of the key proteins in the glial portion of the glutamate-glutamine cycle and may impact on transmitter clearance, transmitter recycling and, as discussed, on the development of the cellular architecture in retina.

Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis.

To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.

Two roles for the leucine-responsive regulatory protein in expression of the alanine catabolic operon (dadAB) in Klebsiella aerogenes.

The lrp gene, which codes for the leucine-responsive regulatory protein (Lrp), was cloned from Klebsiella aerogenes W70. The DNA sequence was determined, and the clone was used to create a disruption of the lrp gene. The lack of functional Lrp led to an increased expression of the alanine catabolic operon (dad) in the absence of the inducer L-alanine but also to a decreased expression of the operon in the presence of L-alanine. Thus, Lrp is both a repressor and activator of dad expression. Lrp is also necessary for glutamate synthase formation but not for the formation of two other enzymes controlled by the nitrogen regulatory (Ntr) system, glutamate dehydrogenase and histidase.

Glutamine synthetase expression and activity are regulated by 3,5,3'-triodo-L-thyronine and hydrocortisone in rat oligodendrocyte cultures.

Glutamine synthetase plays a central role in the detoxification of brain ammonia. Previously, we demonstrated that in vitro glutamine synthetase is expressed by all macroglial cell types and is developmentally regulated in oligodendrocyte lineage. Furthermore, glutamine synthetase is increased in secondary cultures of oligodendrocytes following a 72 h treatment with 30 nM 3,5,3'-triodo-L-thyronine [Baas, D., Bourbeau, D., Sarliève, L. L., Ittel, M. E., Dussault, J. H. and Puymirat, J., Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone. Glia, 1997, 19, 324-332]. Hydrocortisone also increases glutamine synthetase activity after 72 h [Fressinaud, C., Weinrauder, H., Delaunoy, J. P., Tholey, G., Labourdette, G. and Sarliève, L. L., Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors. J. Cell. Physiol., 1991, 149, 459-468]; however, it is still unknown whether these increases in glutamine synthetase expression in oligodendrocytes after 3,5,3'-triodo-L-thyronine and hydrocortisone application are dose- and time-dependent. To further investigate this issue, we measured glutamine synthetase levels by Northern analysis, immunostaining and determination of glutamine synthetase activity after 3,5,3'-triodo-L-thyronine or hydrocortisone stimulation. We find that in rat oligodendrocyte secondary cultures, 3,5,3'-triodo-L-thyronine and hydrocortisone cause a dose- and time-dependent increase in glutamine synthetase mRNA, protein and activity. However, these hormones do not exert an additive or synergistic effect. Because purines, pyrimidines, and certain amino acids necessary for the synthesis of myelin components, are, in part, provided by the glutamine synthetase pathway, 3,5,3'-triodo-L-thyronine effect on myelination development and maturation could be mediated in part, through the glutamine synthetase gene regulation.

The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Comparison of the sensitivities of two Escherichia coli genes to in vivo variation of Lrp concentration.

Transcription of the Escherichia coli genes serA and gltBDF depends on the leucine-responsive regulatory protein, Lrp, and is very much decreased in an lrp mutant. By the use of an Lrp-deficient host and the lrp gene cloned under a plasmid-borne arabinose pBAD promoter, we varied the amount of Lrp present in the cell and showed that both genes were transcribed in proportion to the amount of Lrp synthesized. The affinity of serA for Lrp was four to five times greater than the affinity of gltD. Overproduction of Lrp was lethal to the cell.

Structure and regulation of ferredoxin-dependent glutamase synthase from Arabidopsis thaliana. Cloning of cDNA expression in different tissues of wild-type and gltS mutant strains, and light induction.

Ferredoxin (Fd)-dependent glutamate synthase is present in green leaves, etiolated leaves, shoots and roots of Arabidopsis thaliana (ecotype Columbia). In photosynthetic green leaves and shoots, Fd-dependent glutamate synthase accounts for more than 96% of the total glutamate synthase activity in vitro with the remaining activity derived from an enzyme that uses NADH as the electron donor. In etiolated leaves and roots, Fd-dependent glutamate synthase is 3-4-fold less active than in green leaves, but represents 70-85% of the total glutamate synthase activity in these tissues. Fd-dependent glutamate synthase is detected as a single peptide of 165 kDa on a western blot of green leaf and shoot tissues, and this Fd-dependent glutamate synthase polypeptide is 3-4-fold less abundant in etiolated leaves and roots. In these non-photosynthetic tissues, there is a higher activity of NADH-dependent glutamate synthase. The A. thaliana gltS mutant (strain CS254) contains only 1.7% and 17.5% of the wild-type Fd-dependent glutamate synthase activity in leaves and roots, respectively. Western blots indicate that the Fd-dependent glutamate synthase peptide of 165 kDa is absent from leaves and roots of the gltS mutant. In contrast, NADH-dependent glutamate synthase activity in leaves and roots is unaffected. During illumination of wild-type dark-grown leaves for 72 h, the levels of Fd-dependent glutamate synthase protein and its activity increased threefold to levels equivalent to those in green leaves. In contrast, NADH-dependent glutamate synthase activity decrease twofold during illumination. The complete nucleotide sequence of the complementary DNA for A. thaliana Fd-dependent glutamate synthase has been determined. Analysis of the amino acid sequence deduced from the complete cDNA sequence (5178 bp) has revealed that A. thaliana Fd-dependent glutamate synthase is synthesized as a 1648-amino-acid precursor protein (180090 Da) which consists of a 131-amino-acid transit peptide (14603 Da) and a 1517-amino-acid mature peptide (165487 Da). The A. thaliana Fd-dependent glutamate synthase has a high similarity to maize Fd-dependent glutamate synthase (83%) and to the analogous region of NADH-dependent glutamate synthase (42%) and NADPH-dependent glutamate synthases (40-43%) from different organisms. The A. thaliana Fd-dependent glutamate synthase contains the purF-type glutamine-amido-transfer domain as well as flavin and iron-sulfur-cluster-binding domains. The deduced primary structures of A. thaliana Fd-dependent glutamate synthase and of glutamate synthases from other organisms indicate that Fd-dependent glutamate synthase may have evolved from bacterial NADPH-dependent glutamate synthase. The cDNA hybridized to RNA of about 5.3 kb from different tissues of A. thaliana. A high steady-state level of Fd-dependent glutamate synthase mRNA is found in photosynthetic green leaves and shoots, and roots contain less mRNA for Fd-dependent glutamate synthase. In the gltS mutant, there are twofold and fourfold lower levels of Fd-dependent glutamate synthase mRNA in leaves and roots, respectively, relative to those in wild-type A. thaliana. Under continuous illumination of dark-grown leaves, the Fd-dependent glutamate synthase mRNA is induced twofold to a level equivalent to that in green leaves.

Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli.

Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains. We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp. To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I. C. Blomfield, P. J. Calie, K. J. Eberhardt, M. S. McClain, and B. I. Eisenstein, J. Bacteriol. 175:27-36, 1993) or on a multicopy plasmid. Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG. Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations. beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal. The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B. R. Ernsting, J. W. Denninger, R. M. Blumenthal, and R. G. Matthews, J. Bacteriol. 175:7160-7169, 1993). Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression. The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates. Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level.

Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression.

The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB. The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp. A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position -64 with respect to the gltA transcription start. This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC. Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the -35 region of the gltA promoter. Box II was found to be essential for activation of gltA but not for gltC autoregulation. Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7- to 40-fold under nonactivating conditions and about 2-fold under activating conditions. Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA. gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn.

Mutations in GltC that increase Bacillus subtilis gltA expression.

Mutants with altered forms of GltC, a positive LysR-type regulator of Bacillus subtilis glutamate synthase gene expression, were isolated. The mutant GltC proteins stimulated expression from the wild-type gltA promoter region 1.5- to 2.0-fold and from mutant promoter regions up to 80-fold. Moreover, expression of gltA became much less dependent on a nitrogen source-associated signal.

Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation.

Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.

Glutamate synthase from Bacillus subtilis PCI 219.

Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.

Polarity in the glnA operon: suppression of the reg- phenotype by rho mutations.

To determine the ability of mutations in glnA, the gene for glutamine synthetase (GS), to regulate nitrogen assimilatory enzymes, we assayed histidase and GS in 34 glnA (Gln(-)) strains. Twenty-five glnA mutants were RegC, synthesizing high levels of histidase regardless of the availability of nitrogen, and nine were Reg(-), synthesizing low levels of histidase in medium containing either limiting or excess ammonia. rho mutations were introduced into strains containing glnA point mutations or insertions in glnA, glnL, glnG, or glnF. The Reg(-) phenotype of strains with glnA point mutations, but not those with glnA or glnF insertions, was altered by the presence of rho, suggesting that glnA (Reg(-)) mutations are polar and exert their phenotype by decreasing expression of glnL and glnG. Consistent with this view, no GS protein was detected by two-dimensional gel electrophoresis in glnA (Reg(-)) rho(+) or glnA (Reg(-)) rho double mutants, whereas GS protein was detected in cells of 10 of 11 glnA (RegC) strains. Since glnA (Reg(-)) rho double mutants synthesize constitutive levels of histidase, GS protein is not necessary for full expression of histidase. Mu d1 insertions in glnL, but not those in glnG, responded to the presence of a rho allele, presumably owing to elevated transcription into glnG from the Mu d1 prophage. Our results suggest that glnA (Reg(-)) alleles are polar mutations, and a rho-dependent termination site down-stream is postulated as the basis for the polar phenomenon. The data also indicate that, under some circumstances, a significant portion of glnL and glnG transcription is initiated at the glnA promoter.

Mapping and characterization of the hisD-unlinked nif mutants in Klebsiella pneumoniae.

Two hisD-unlinked genes NifC5 and NifC7 are mapped in the chromosome of K. pneumoniae. The sequence of NifC5 and NifC7 is suggested as NifC5--gltB--NifC7--argG. The P1 infected E. coli lysate can transduce the mutant C-7 to be Nif+ transductant, yet fails to transduce the hisD-linked nif mutants to be Nif+ ones. This indicates that the gene encoding C-7 is not the structural gene of nitrogen fixation and is present in E. coli. It is actually a gene specifying the glutamate synthetase. SDS electrophoresis shows the marked low content of nitrogen reductase and immunoelectrophoretic test reveals the reduced amount of both nitrogenase and nitrogen reductase in the mutant cells.