Fasting-induced changes in ECL cell gene expression.

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.

Histidine dissimilation in Streptomyces coelicolor.

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.

Regulation of hut enzymes and intracellular protease activities in Vibrio alginolyticus hut mutants.

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.

The effect of nitrogen limitation on catabolite repression of amidase, histidase and urocanase in Pseudomonas aeruginosa.

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.

Expression of the hut operons of Salmonella typhimurium in Klebsiella aerogenes and in Escherichia coli.

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.

Involvement of threonine dehydratase in biosynthesis of the alpha-ketobutyrate prosthetic group of urocanase.

Seventeen mutants of Pseudomonas putida that were unable to grow on threonine as nitrogen source owing to a lack of threonine dehydratase were isolated, and all were found to be unable to synthesize active urocanase. Spontaneous revertants selected for urocanase production concomitantly regained threonine dehydratase. Mutants that were unable to utilize urocanate as carbon source were also isolated, and these were defective in urocanase formation but were normal in threonine dehydratase levels. Since alpha-ketobutyrate is the prosthetic group for urocanase, these results are consistent with the proposal that threonine dehydratase is necessary for urocanase prosthetic group biosynthesis. However, the lack of urocanase activity in threonine dehydratase-negative mutants was shown not to be the result of reduced levels of endogenous free alpha-ketobutyrate, nor to the participation of threonine dehydratase in the initiation of urocanase biosynthesis through the conversion of threonyl-tRNA(Thr) to alpha-ketobutyryl-tRNA(Thr). Other alternatives for the participation of threonine dehydratase in urocanase biosynthesis are discussed.

Effect of temperature on histidine ammonia-lyase from a psychrophile, Pseudomonas putida.

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 control of the enzymes degrading histidine and related imidazolyl derivates in Pseudomonas testosteroni.

1. The induction of the enzymes for the degradation of l-histidine, imidazolylpropionate and imidazolyl-l-lactate in Pseudomonas testosteroni was investigated. 2. The activities of histidine ammonia-lyase, histidine-2-oxoglutarate aminotransferase and urocanase are consistent with these enzymes being subject to co-ordinate control under most growth conditions. However, a further regulatory mechanism may be superimposed for histidase alone under conditions where degradation of histidine must take place for growth to occur. 3. Experiments with a urocanase(-) mutant show that urocanate is an inducer for the enzymes given above and also for N-formiminoglutamate hydrolyase and N-formylglutamate hydrolase. 4. N-Formiminoglutamate hydrolase and N-formylglutamate hydrolase are also induced by their substrates, and it is suggested that these two enzymes may be different gene products from those expressed in the presence of urocanate. 5. Induction of the enzyme system for the oxidation of imidazolylpropionate is dependent on exposure of cells to this compound.