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Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.

The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene. The E. coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC(E) is 305 amino acids, the same as for NAC(K). A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC(E) by using methods established for NAC(K) failed, and NAC(E) appears to be degraded with a half-life at 30 degrees C as short as 15 min during inhibition of protein synthesis.

The effect of glucagon and hydrocortisone was investigated to understand the mechanism of induction of hepatic histidase gene. In this study, glucagon (0.6 mg/100 g body wt/d) was injected to rats fed 10% casein diet. After 3 h of the last injection, histidase activity and amount of enzyme were induced by 3 fold and histidase mRNA concentration by 6 fold. Injection of hydrocortisone (2 mg/100 g body wt/d) increased 100% histidase activity and mRNA concentration and by 150% the amount of enzyme after 3 h of the last injection. These results indicate that glucagon is a better inductor of histidase gene expression than hydrocortisone. Another purpose of the study was to evaluate if a protein-free/high carbohydrate diet could reverse the induction of Hal expression produced by a high protein diet. Hal activity, amount of enzyme and mRNA concentration was repressed by 68, 88 and 95% respectively by a protein-free/high carbohydrate diet. Injection of glucagon reversed partially the effect of a high carbohydrate diet, however, injection of hydrocortisone under the concentration used in these experiments did not reverse the effect of a high carbohydrate diet. These results support the evidence that hepatic histidase gene expression is probably regulated transcriptionally by hormones.

Histidase (EC 4.3.1.3) is a cytosolic enzyme that catalyzes the nonoxidative deamination of histidine to urocanic acid. Histidinemia, resulting from reduced histidase activity as reported in Cambridge stock his/his mice and in humans, is the most frequent inborn metabolic error in Japan. The histidase chromosomal gene (HAL) was isolated from a lambda EMBL-3 human genomic library using the human histidase cDNA as a probe. Restriction mapping and Southern blot analysis of the isolated clones reveal a single-copy gene spanning approximately 25 kb and consisting of 21 exons. Exon 1 encodes only 5' untranslated sequence of liver histidase mRNA, with protein coding beginning in exon 2. A rarely observed 5' GC, similar to that reported in the human P-450 (SCC) gene, is present in intron 20. All other splicing junctions adhere to the canonical GT/AG rule. A TATA box sequence is located 25 bp upstream of the liver histidase transcription initiation site determined by S1 nuclease protection analysis. Several liver- and epidermis-specific transcription factor binding sites, including C/EBP, NFIL6, HNF5, AP2/KER1, MNF, and others, are also identified in the 5' flanking region. Consistent with the hepatic and epidermal expression of histidase, this finding suggests that histidase transcription may be regulated by these factors. We further identify a polymorphism (A to G transition) in the histidase coding region of exon 16. The human histidase genomic structure presented here should facilitate the molecular investigation of symptomatic and asymptomatic forms of histidinemia.

Expression of the Bacillus subtilis hut operon is subject to regulation by catabolite repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify two cis-acting sites involved in catabolite repression. The hutOCR1 operator site lies immediately downstream of the hut promoter and weakly regulates hut expression in response to catabolite repression. The downstream hutOCR2 operator site lies within the hutP gene, between positions +203 and +216, and is required for wild-type levels of catabolite repression. Both the hutOCR1 and hutOCR2 operators have sequence similarity to the sites which mediate catabolite repression of several other B. subtilis genes. Two mutations which relieve catabolite repression of hut expression were found to alter the nucleotide sequence of the hutOCR2 operator. Catabolite repression of hut expression was partially relieved in strains containing the ccpA mutation but not in strains containing either the pai or hpr mutation.

The first enzyme of the Bacillus subtilis histidine-degradative (hut) pathway, histidase, was expressed at higher levels during the onset of the stationary growth phase in nutrient sporulation medium in early-blocked sporulation mutants (spo0A) than in wild-type strains. Histidase expression was also elevated in spo0A mutant cultures compared with wild-type cultures during the logarithmic growth phase in minimal medium containing slowly metabolized carbon sources. Histidase expression was not derepressed in spo0A abrB mutant cultures under these growth conditions, suggesting that the AbrB protein is responsible for the derepression of histidase synthesis seen in spo0A mutant cultures. spo0A mutants contain higher levels of the AbrB protein than do wild-type strains because the Spo0A protein represses AbrB expression. A direct correlation between the levels of abrB transcription and histidase expression was found in spo0A mutant cultures. The hutOCR2 operator, which is required for wild-type regulation of hut expression by catabolite repression, was also required for AbrB-dependent derepression of hut expression in spo0A mutants. Purified AbrB protein bound to the hutOCR2 operator in vitro, suggesting that AbrB protein alters hut expression by competing with the hut catabolite repressor protein for binding to the hutOCR2 site. During the logarithmic growth phase in media containing slowly metabolized carbon sources, the expression of several other enzymes subject to catabolite repression was elevated in spo0A mutants but not in spo0A abrB mutants. This suggests that the AbrB protein acts as a global modulator of catabolite repression during carbon-limited growth.

Histidine ammonia-lyase (HAL) from Pseudomonas putida PRS1 contains a catalytically important electrophilic center reported to be dehydroalanine. Little is known about the origin of this group or its linkage to the protein. To initiate structural studies on this enzyme, P. putida HAL was purified from an Escherichia coli high-expression clone in which the HAL gene (hutH) was under the control of the lambda PL promoter on a plasmid vector. In this clone from 6 to 10% of the soluble cell protein after heat induction was HAL and approximately 200 mg of 95% pure HAL could be obtained from 120 g wet weight of cells in a 40 to 60% yield. The overexpressed protein was identical to P. putida HAL in native molecular weight (220 kDa), subunit composition (four identical subunits of 53 kDa each), affinity for substrate (L-histidine Km of 5.3 mM at pH 9.0), and its sensitivity to inactivation by cyanide and bisulfite. The N-terminal amino acid sequence was in agreement with the DNA-predicted sequence, indicating proper translational initiation. These features make this enzyme an appropriate candidate for protein structure investigations regarding the nature of the electrophilic center and its association with the protein.

In Klebsiella aerogenes, the formation of a large number of enzymes responds to the quality and quantity of the nitrogen source provided in the growth medium, and this regulation requires the action of the nitrogen regulatory (NTR) system in every case known. Nitrogen regulation of several operons requires not only the NTR system, but also NAC, the product of the nac gene, raising the question of whether the role of NAC is to activate operons directly or by modifying the specificity of the NTR system. We isolated an insertion of the transposon Tn5tac1 which puts nac gene expression under the control of the IPTG-inducible tac promoter rather than the nitrogen-responsive nac promoter. When IPTG was present, cells carrying the tac-nac fusion activated NAC-dependent operons and repressed NAC-repressible operons independent of the nitrogen supply and even in the absence of an active NTR system. Thus, NAC is sufficient to regulate operons like hut (encoding histidase) and gdh (encoding glutamate dehydrogenase), confirming the model that the NTR system activates nac expression and NAC activates hut and represses gdh. Activation of urease formation occurred at a lower level of NAC than that required for glutamate dehydrogenase repression, and activation of histidase formation required still more NAC.

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.

The first enzymes of the histidine (hut) and proline degradative pathways, histidase and proline oxidase, could not be induced in Bacillus subtilis cells growing in glucose minimal medium containing a mixture of 16 amino acids. Addition of the 16-amino-acid mixture to induced wild-type cells growing in citrate minimal medium repressed histidase synthesis 25- to 250-fold and proline oxidase synthesis 16-fold. A strain containing a transcriptional fusion of the hut promoter to the beta-galactosidase gene was isolated from a library of Tn917-lacZ transpositions. Examination of histidase and beta-galactosidase expression in extracts of a hut-lacZ fusion strain grown in various media showed that induction, catabolite repression, and amino acid repression of the hut operon were mediated at the level of transcription. This result was confirmed by measurement of the steady-state level of hut RNA in cells grown in various media. Since amino acid repression was not defective in B. subtilis mutants deficient in nitrogen regulation of glutamine synthetase and catabolite repression, amino acid repression appears to be mediated by a system that functions independently of these regulatory systems.

The effects of benzonal on the course of neonatal hemolytic disease due to the Rhesus factor-conflict was studied in comparison with that of phenobarbital. Dynamic follow-up of infants in the early neonatal period showed benzonal to produce a more pronounced hypobilirubinemic effect which was manifested as a prompter disappearance of skin jaundice and lower percentages of complications. By depressing the activity of organospecific enzymes and lowering the serum biliary acid levels, benzonal promotes normalization of the metabolic shifts present in neonatal hemolytic disease. The findings make it possible to recommend the new inductor of microsomal liver enzymes benzonal as part of the combined therapy of neonatal hemolytic disease.

Synthesis of rat liver histidase is under multihormonal control; estrogen, glucocorticoid, and glucagon, via cAMP, induce this enzyme. By means of in vivo [3H]leucine incorporation into immunoprecipitated histidase, relative to that incorporated into total soluble protein, we have now demonstrated that de novo hepatic histidase synthesis, as well as catalytic activity, is selectively increased following hypophysectomy. Treatment of hypophysectomized rats with physiological levels of triiodothyronine (T3) diminished histidase synthetic rates and catalytic activities to normal levels, despite concomitant elevation in total soluble protein synthesis. Thyrotoxic doses of T3 further reduced histidase synthesis to barely measurable rates. Since thyroid hormone is under pituitary regulation, this hormone may be primary in the hypophyseal suppression of histidase. Estrogen does not induce hepatic histidase in the hypophysectomized rat. However, administration of the histidase suppressor, T3, or prolactin, which in itself has no effect on this enzyme, was ineffective in reinstating estrogen inducibility of histidase in the hypophysectomized animal. Thus, some as yet unknown hypophyseal agent is required for estrogenic inducibility of this liver enzyme.

The mechanisms by which estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate (cAMP), regulators which participate in the postnatal development of rat liver histidase, elevate the catalytic activity of this enzyme have been explored. A monospecific antibody against homogeneously purified preparations of rat liver histidase has been elaborated in the goat. Employing this antibody in immunotitration experiments, it has been demonstrated that the elevations of hepatic histidase activity elicited by administration in vivo of estradiol-17beta, cortisol acetate, glucagon, and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) are paralleled, in each instance, by equivalent increments in immunoprecipitable histidase protein. Following administration of each of the three hormones and dibutyryl cAMP, rates of [14C]leucine incorporation in vivo into rat liver histidase, isolated by immunoprecipitation, relative to incorporation rates into total soluble hepatic protein, increase in magnitudes which are comparable to increases in enzyme amount and catalytic activity. It is thus inferred that estrogen, glucocorticoids, and glucagon, via cAMP, each regulate rat liver histidase development at specific postnatal stages by inducing increases in histidase biosynthetic rates.

In Klebsiella aerogenes but not in Salmonella typhimurium glutamine synthetase can function during nitrogen-limited growth to increase the rate of synthesis of histidase from the hut genes of S. typhimurium 15-59 (hutS. 15-59). Formation of proline oxidase is also not increased in nitrogen-limited cultures of S. typhimurium. However, in hybrid strains of Escherichia coli or K. aerogenes, the glutamine synthetase of S. typhimurium activates synthesis of histidase from the hutS. 15-59 genes. Apparently, glutamine synthetase is necessary but not sufficient for activation of transcription of the hut genes; another factor must also be present. This factor is active in both K. aerogenes and E. coli but is missing or altered in S. typhimurium.

A pleiotropic mutant of Bacillus subtilis was isolated which overproduced in the presence of glucose several enzymes whose synthesis is subject to glucose catabolite repression. Examination of intracellular metabolites suggested that the mutation may have resulted in a defect in glycolysis, increasing phosphoenolpyruvate and decreasing pyruvate, 2-ketoglutarate, and oxaloacetate.

We have partially characterized the biochemical parameters of glutamine synthetase from Klebsiella pneumoniae and have shown that the differential affinity of adenylylated and unadenylylated glutamine synthetase for adenosine diphosphate provides a convenient means of determining the adenylylation state. Using this assay procedure, we examined the relationship between the adenylylation state and the expression of other genes involved in nitrogen assimilation. We observed no correlation between the adenylylation state and the expression of histidase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, and urease in aerobic cultures.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.

Inducibility of histidase (histidine ammonia-lyase, EC 4.3.1.3) in Pseudomonas putida and Pseudomonas aeruginosa was observed to be strongly affected by succinate-provoked catabolite repression, but this did not occur as a consequence of reduced intracellular cyclic adenosine 3',5'-monophosphate levels, and repression could not be alleviated by exogenously added cyclic adenosine 3,'5'-monophosphate. Milder repression of histidase by lactate was also not reversed by the addition of cyclic adenosine 3',5'-monophosphate. These results, along with data showing intracellular cyclic adenosine 3',5'-monophosphate levels remained essentially constant during growth on such diverse carbon sources as histidine, acetamide, glucose, and succinate, indicated that catabolite repression of histidase synthesis by efficient carbon sources was not mediated through variations in internal cyclic adenosine 3,'5'-monophosphate.

Regulation of the expression of the histidase coded by hutk of Klebsiella aerogenes in Salmonella typhimurium and in Escherichia coli and of the expression of the histidase coded by huts of S. typhimurium in E. coli was investigated. The hutk histidase was found to be sensitive to catabolite repression in K. aerogenes and in E. coli, but insensitive to catabolite repression in S. typhimurium; huts histidase has previously been shown to be catabolite sensitive in all three organisms. The expression of both hutk and huts histidase in E. coli was activated by nitrogen starvation. Apparently, the glutamine synthetase of E. coli may activate the formation of some glutamate- and ammonia-producing enzymes.

A mutation of Salmonella typhimurium was obtained that results in the failure of cells to synthesize the enzyme l-histidine ammonia-lyase (histidase). The mutation mapped within the hutH gene and in merodiploid strains was dominant over the wild-type allele. Extracts from cells bearing the trans-dominant histidase-negative allele were shown to contain material that reacts immunologically with antiserum against purified wild-type histidase. It is proposed that the trans-dominant allele results in the synthesis of defective histidase subunits that can combine with, and partially inactivate, wild-type histidase subunits. This subunit mixing presumably does occur, as the enzyme synthesized in a hybrid merodiploid strain is abnormally heat sensitive.

Repeated administration of ethanol into animals within 14 and 21 days decreased or completely abolished the inducing effect of ethanol on the tyrosine aminotransferase activity (TAT) in liver tissue. At the same time, the inducing effect of hydrocortisone was maintained although at the lower level as compared with the intact animals. Chronic poisoning of the animals with CCl4 within 4 weeks inhibited the inducing effect. As compared with controls the inducing effect of hydrocortisone was distinctly lower in the animals poisoned with CCl4.

In Serratia marcescens, the mutation responsible for triazolealanine (TRA) resistance was transferred from a TRA-resistant mutant to a urocanase-less mutant by PS20-mediated transduction. The two crosses were performed using as donors two TRA-resistant mutants, whose phenotypes included increased levels of histidine-biosynthetic enzymes and feedback-insensitive phosphoribosyltransferase. In one cross, TRA-resistant transductants were urocanase-less mutants having only increased levels of the enzymes and barely detectable levels of urocanic acid. In the other cross, the transductants were urocanase-less mutants having both phenotypes of the donor, and most produced high concentrations (10.5 mg/ml) of urocanic acid.

An attempt was made to determine whether sporulation and inducible enzyme synthesis in Bacillus subtilis are controlled by the same mechanism of catabolite repression. By the use of a thymine-requiring strain, it has been shown that, whereas sporulation remained repressed unless chromosome replication proceeded to completion, the induction of the enzymes histidase, sucrase, and alpha-glucosidase proceeded quite normally in the absence of continued deoxyribonucleic acid synthesis. It is concluded that the mechanism for overcoming the repression of sporulation differs qualitatively from that involved in overcoming the repression of inducible enzyme synthesis. Attempts to isolate pleiotropic mutants that would provide additional support for this contention were unsuccessful. A pleiotropic mutant deficient in phosphoenolpyruvate-dependent phosphotransferase activity sporulated quite well, whereas a mutant presumed deficient in glutamate synthetase sporulated poorly under all conditions.

Pseudomonas putida was able to grow at 0 C in a complex medium containing l-histidine and to synthesize histidine ammonia-lyase and urocanase. The activity of the former enzyme was assessed between -10 and 60 C in cells and in cell extracts. Activity was maximal from 20 to 35 C. Below 20 C, activity decreased with temperature but, significantly, the enzyme exhibited 30% of its maximal activity at 1.5 C. The temperature response was similar in both intact cells and cell extracts, which indicated that the cell membrane did not significantly limit the entry of histidine at low temperature. Above and below the maximal temperature range, the reduced activity was not caused by irreversible inactivation, as shown by preincubation experiments. Also, when the temperature was rapidly changed from 60 to 30 C during an assay, the reaction rate increased abruptly to the full 30 C activity without a lag. This demonstrated the rapid reversibility of inactivation. The apparent Michaelis constant increased with temperature. As the substrate concentration was decreased, the enzyme activity became less dependent on temperature. The efficiency of substrate entry and catalysis near 0 C are factors in the ability of this facultative psychrophile to grow in a histidine medium at 0 C.

The normal hut (histidine utilization) operons, as well as those with mutations affecting the regulation of their expression, of Salmonella typhimurium were introduced on an F' episome into cells of S. typhimurium and Klebsiella aerogenes whose chromosomal hut genes had been deleted and into cells of Escherichia coli, whose chromosome does not carry hut genes. The episomal hut operons respond in a manner very similar to induction and catabolite repression in all three organisms. The small differences found reflect both different abilities to take up inducers from the medium and different degrees of catabolite repression exerted by glucose.

In Pseudomonas aeruginosa, the synthesis of histidase, urocanase and amidase is severly repressed when succinate is added to a culture growing in pyruvate + ammonium salts medium. When growth is nitrogen-limited, catabolite repression by succinate of histidase and urocanase synthesis does not occur but succinate repression of amidase synthesis persists. Amidase synthesis is not regulated in the same way as histidase synthesis by the availability of other nitrogen compounds for growth. Growth of P. aeruginosa strain PACI in succinate + histidine media is nitrogen-limited since this strain is defective in a histidine transport system. When methyl-ammonium chloride is added to succinate + histidine media, growth inhibition occurs. Mutants isolated from succinate + histidine + methylammonium chloride plates were found to be resistant to catabolite repression by succinate even in ammonium salts media. It is suggested that the hut genes of P. aeruginosa may be regulated in the same way as in Klebsiella aerogenes, by induction by urocanate and activation by either the cyclic AMP-dependent activator protein or by glutamine synthetase.

The synthesis of histidase occurs only in free polyribosomes. The relative content of histidase synthesizing polyribosomes in rat liver, in Zajdela ascites hepatoma cells and in the liver of tumor-bearing rats is equal to 1.35%, 0.11% and 0.57%, respectively (of the total amount of free polyribosomes). It was found that hepatoma cell sap has an inhibitory effect on the synthesis of proteins in the cell-free system reconstructed from polyribosomes and cell sap of control rats.

Netropsin, a polypeptide antibiotic which binds specifically to adenylate-thymidylate-rich regions of deoxyribonucleic acid, inhibitis sporulation at about stage II, but does not inhibit growth of Bacillys subtilis. An analysis of the sporulation-associated enzymes aconitase, alkaline phosphatase, and glucose dehydrogenase revealed that their rates of expression were not affected by the presence of the antibiotic. The derepression of histidase, a vegetatively induced enzyme was stimulated by netropsin. Oxygen utilization by the cells during sporulation was not effected nor was spore germination prevented by the drug. Netropsin, however, did prevent the formation of dipicolinic acid. These and earlier results suggest that netropsin may be affecting the transcription of only select sporulation genes that are particularly rich in adenylate-thymidylate base pairs.

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.

In Klebsiella aerogenes, the product of a his-linked gene, nac, appears to play a crucial role in tying the synthesis of enzymes activated or repressed by ammonia deprivation, such as histidase and glutamate dehydrogenase, to the known regulators of nitrogen assimilation, the products of glnG and glnF.

Urease was purified 24-fold from extracts of Klebsiella aerogenes. The enzyme has a molecular weight of 230,000 as determined by gel filtration, is highly substrate specific, and has a Km for urea of 0.7 mM. A mutant strain lacking urease was isolated; it failed to grow with urea as the sole source of nitrogen but did grow on media containing other nitrogen sources such as ammonia, histidine, or arginine. Urease was present at a high level when the cells were starved for nitrogen; its synthesis was repressed when the external ammonia concentration was high. Formation of urease did not require induction by urea and was not subject to catabolite repression. Its synthesis was controlled by glutamine synthetase. Mutants lacking glutamine synthetase failed to produce urease, and mutants forming glutamine synthetase at a high constitutive level also formed urease constitutively. Thus, the formation of urease is regulated like that of other enzymes of K. aerogenes capable of supplying the cell with ammonia or glutamate.

Studies on the induction of histidase were made with normal and nitrofurantoin resistant strains of Vibrio el tor. Nitrofurantoin resistant strains showed decreased level of induction in comparison to normal Vibrio el tor. The effect of different inhibitors like actinomycin D, chloramphenicol, tetracycline, nitrofurantoin and rifampicin on histidase induction was also studied. The mechanism of inhibition caused by the antibiotics is discussed.

A growth technique that allows strains of Streptomyces coelicolor to grow dispersed in defined liquid medium has been devised and used to determine the pathway of histidine degradation by S. coelicolor. Enzymic, chromatographic and stoichiometric analyses indicated that histidine is dissimilated via N-formyl-L-glutamic acid. The enzymes for histidine utilization (hut) are induced when histidine or urocanate is included in the culture medium. Biochemical evidence suggested that urocanate, or a further metabolite, is the physiological inducer. Three hut mutants were isolated and characterized. Two of the mutants exhibit an uninducible phenotype, whereas the third mutant appears to be defective in the structural gene for formiminoglutamate iminohydrolase. Haploid recombinant analysis was employed to locate all three mutations in the left empty region of the chromosomal map.

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.

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA. The product of glnG is essential for normal regulation of the synthesis of glutamine synthetase and histidase. We analyzed two mutations which affected the regulation of the formation of these enzymes and appeared to be outside glnG. The results of our studies suggest the presence in K. aerogenes of at least two regulatory genes located in the glnA region, namely, glnG and another gene, the transcription of which can be initiated at the promoter of glnA.

Inbred strains of mice fall into two groups with respect to their liver histidase activity levels, high strains having approximately twice as much activity as low strains. Analysis of the F1, F2, and backcross progeny of the mating of a high activity strain (C57BL/6J) and a low activity strain (C3H/HeJ) indicates that the difference between the strains is determined by a single genetic locus with two alleles exhibiting additive inheritance. No differences with respect to various physical and kinetic parameters were found in studies of partially purified histidase from both strains. Quantitation of the amount of enzyme present by immunotitration showed that the amount of enzyme antigen is proportional to the level of enzyme activity in the two strains. Measurements of the relative rates of histidase synthesis by combined radiochemical and immunological techniques showed that the relative rate of synthesis was closely correlated with the amount of enzyme present. Rates of enzyme degradation in the two strains, measured by recovery of activity after irreversible inhibition with nitromethane, were the same.

In contrast to wild-type cells, the Bacillus subtilis mutant SF109 that lacks the active 2-ketoglutarate dehydrogenase enzymatic complex is unable to increase the specific activity of two enzymes subject to glucose catabolite repression, aconitase and histidase, during limitation of growth by glucose. Examination of the intracellular metabolite pools in the mutant and wild-type cells grown in excess and limiting glucose medium showed that the complete derepression of aconitase and histidase could be correlated with the decrease in the intracellular concentration of 2-ketoglutarate. The complete repression of aconitase that occurred in wild-type and mutant cells could be correlated with a high intracellular concentration of 2-ketoglutarate.

The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds. The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product. Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter). In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis. The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression.

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.

The production of alkaline protease, collagenase and histidine utilization (Hut) enzymes by Vibrio alginolyticus wild-type, hutH1 and hutU1 strains was investigated. Alkaline protease synthesis was stimulated by histidine and urocanic acid in the wild-type and hutU1 strains. The hutH1 mutant alkaline protease production was stimulated by urocanic acid and not by histidine. The Hut enzymes in the wild-type strain were coordinately induced by histidine. Urocanase and formimino-hydrolase were induced by histidine in the hutH1 mutant which lacked histidase and was not able to convert histidine to urocanic acid. Collagenase production in peptone medium was inhibited in the hut mutants. It is concluded that in V. alginolyticus urocanic acid regulates alkaline protease synthesis but that the Hut enzymes are induced by histidine. The involvement of the Hut genetic system in the regulation of alkaline protease and collagenase synthesis is discussed.

Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase.

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.

Mutants of Klebsiella aerogenes containing genetic fusions of glnA to lacZ were isolated by using Mu dl (lac, bla) bacteriophage and a Mu Kmr helper phage with the host range of bacteriophage P1. Synthesis of beta-galactosidase in these strains is regulated in response to nitrogen metabolites and regulatory gln loci and is rendered constitutive by a mutation in the linked glnL gene. Complementation studies indicated that glnL is a separate locus from glnA and glnG and that insertions in glnA are partially polar on glnL expression. These results support the hypothesis that glnA, glnL, and glnG are organized in an operon with multiple promoters.