Über die Phycobionten der SektionCystophora vonChaenotheca,insbesondereDictyochloropsis splendida undTrebouxia simplex,spec. nova

Chaenotheca brunneola is lichenized withDictyochloropsis splendida. Until now this member of theChlorococcales has been observed only twice and free-living. It reproduces by motionless daughter cells in the lichen thallus, but mainly by zoospores in the free-living state and under favourable conditions. Zoospore development has not been observed before and apparently depends on storage of starch which is repressed in the lichenized state. During zoospore activity this storage material is consumed. Zoospores possess two flagella which are inserted rather far apart from each other, one pulsating vacuole and a cup-shaped, not latticed chloroplast.Chaenotheca phaeocephala var.alpina, Ch. melanophaea andCh. chrysocephala are lichenized withTrebouxia. The phycobiont ofCh. chrysocephala, Trebouxia simplex, sp. n., differs from other sibs of the genus by the relative small size of the cells, the less marked differentiation of the chloroplast and certain details of cell division.

     

The function of nickel on the urease activity of the lichen Evernia prunastri

The lichen Evernia prunastri synthesizes urease (EC 3.5.1.5) as a response to urea supplied in the medium. Nickel is required to produce the active protein. In addition, nickel behaves as an activator of the enzyme (K_a = 8.1 μM) at low concentrations whereas it is shown to be a competitive inhibitor of urease when it is supplied at concentrations higher than 40 μM (K_i = 50 μM).     

The regulation of urease activity in Aspergillus nidulans

Aspergillus nidulans can utilize urea as a sole source of nitrogen but not as a carbon source. Urea is degraded by a urease. Mutation at any one of three genes, ureB, ureC, and ureD, may result in deficient urease activity. The ureB gene is closely linked to ureA, the structural gene for the urea transport protein. The heat lability of a ureB ^– revertant strain, intragenic complementation tests, and the linkage of ureB to ureA suggest that ureB is the urease structural gene. The ureD gene is probably involved in the synthesis or incorporation of a nickel cofactor essential for urease activity. The function of the ureC gene is not known. Urease is not induced but is subject to nitrogen regulation. The urease activities of ammonium-derepressed mutants show that the effector of nitrogen regulation is more likely to be glutamine than ammonium. When glutamine is present in the medium, urease appears to be inactivated by some means which does not involve a newly synthesized protease or a direct interaction between glutamine and urease.     

Construction and characterization of a full-length cDNA library from mycobiont of <Emphasis Type="Italic">Endocarpon pusillum</Emphasis> (lichen-forming Ascomycota)

The first full-length cDNA library for lichenized fungi was constructed from cultured mycobiont of the arid desert lichen Endocarpon pusillum. Based on small-scale sequencing results, 111 genes of the lichenized fungi were identified for the first time, among which 11 genes shared no homology with any known fungal genes. Real-time PCR showed that the size of the mycobiont genome is 39.13 Mb and the copy number of ribosomal RNA gene repeat units is 43. The results of this study will be valuable for the ongoing lichen genome-sequencing project and the large-scale identification of functional genes from lichenized fungi.     

Studies on cytochromes of lichenized fungi under optimized culture conditions

Cytochromes are membrane-bound hemoproteins responsible for the generation of ATP via the electron transport system to fuel the metabolic processes of the organism for their growth. This study reports the properties of cytochromes present in the isolated lichenized fungi of the cultured lichen Usnea ghattensis under optimized conditions. The fungal partner of the cultured lichen Usnea ghattensis contains a, b and c types of cytochromes. The concentrations of a, b and c type cytochromes were found to be significantly high (0.0967, 0.0900, and 0.1030 mM/mg protein, respectively) in the isolated fungal symbiont of cultured lichen grown in malt-yeast extract medium supplemented with 0.01 mol/l sucrose and 0.01 mol/l polyethylglycol. The results suggest that supplementation of additional carbon sources may play a role in optimizing the growth via activating the cytochrome respiratory system in lichenized fungi.     

Mechanisms of regulation of urease biosynthesis in Proteus rettgeri

Urease of Proteus rettgeri is an inducible enzyme synthesized specifically in the presence of urea; urea analogues did not act as inducers. Once initiated, the biosynthesis of the enzyme proceeded as a constant fraction of the total protein formed. The rate of urease formation was affected by the carbon source used. In comparison with glycerol, glucose inhibited enzyme synthesis. The addition of ammonium ions to the inducing medium also decreased the rate of urease biosynthesis, and when ammonium ions were present urease activity and urea transport across the cell membrane were inhibited. A kinetic analysis of urease inhibition by ammonium ions, by use of a partially purified preparation of urease, showed that it was a competitive inhibition.     

The activation of urease

1. It has been shown that the activity of solutions of twice recrystallized urease is reversibly increased by moderate heating and reversibly decreased by storage in the cold, even in the frozen state. 2. Crude extracts of jack bean meal containing potent urease undergo this same type of reversible activation by heating and inactivation by cooling. Dilution has the same potentiating effect on the activity as moderate heating. As much as a fivefold increase in activity can be obtained when a sample previously inactivated by storage for 24 hours at -10 degrees C. is heated for 5 minutes at 60 degrees C. 3. Solutions of crystalline urease protected by serum albumin and preserved in the cold give a constant "potential" activity over a period of more than 30 days if heated 5 minutes at 60 degrees C. before assay. 4. The data presented have been interpreted to mean that an association between urease molecules (or between urease and other proteins) might occur, resulting in inactivation of the enzyme which would be reversed on dissociation. 5. It has been postulated that the same forces are responsible for the reversible inactivation brought about by standing at temperatures above or below the freezing point.     

Patterns of urease synthesis in developing soybeans

An examination of in vivo polysome-bound activity indicates that soybean (Glycine max, cv. Prize) seed urease is synthesized on large polysomes (n ≥ 15). In vitro urease synthesis is directed by a large RNA (3,000-3,300 nucleotides). Urease synthesis occurs throughout the normal protein biosynthetic phase of the developing seed. Surprisingly, the activity/antigen ratios of urease increase throughout development. Urease appears to be in a more highly polymerized state in mature beans versus beans in early development.     

Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

Nickel availability and urease expression in Proteus mirabilis

Cells of Proteus mirabilis, previously grown in nutrient broth (NB), exhibited an increase in urease activity during subsequent incubation in mineral medium even when protein biosynthesis was inhibited. During growth in NB, degradation of amino acids obviously led to the formation of nickel-complexing metabolites, and nickel ions were therefore inavailable for maximal expression of enzymatically active urease; this inhibition of urcase biosynthesis was overcome by the addition of nickel to the growth medium, and also by added glucose. Experiments concerning the incorporation of radioactive nickel into urease finally indicated that the observed increase in urease activity was caused by posttranslational insertion of nickel into preformed apourease.     

A soybean seed urease-null produces urease in cell culture

Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from `wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize).     

Repression of urease biosynthesis in Neurospora crassa by ammonium ions]

The regulation of the synthesis of the enzyme urease (urea amido hydrolase E.C. 3.5.1.5.) in Neurospora crassa was investigated. The biosynthesis of urease is repressed by ammonium ions. Under ammonium excess conditions the specific activity of urease decreases from 0.980 to 0.180 mumoles NH3/min/mg protein. By addition of cycloheximide it was shown that ammonia influences the synthesis of this enzyme. Enzyme induction by the substrate could be excluded. Even under the conditions of highest repression a specific activity of urease of 0.180 mumoles NH3/min/mg protein was measured. Possible causes of this constitutive enzyme level are discussed.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Characterization of urease from Sporosarcina ureae

Alkaline stable (pH 7.75–12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably active with a specific activity of greater than 9300 μmol urea degraded min^-1 mg protein^-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to posses a homopolymeric urease containing 1 nickel per subunit [M _r=65000], the S. ureae enzyme is comprised of three subunits [apparent M _r=63100 (α), 14500 (β), and 8500 (γ)] in an estimated ∝βγ stoichiometry and contains 2.1±0.6 nickel ions per ∝βγ unit as measured by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment, dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells.     

Slow adaptive changes in urease levels of tobacco cells cultured on urea and other nitrogen sources

Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4-fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 × 10⁻⁵, then multiplied 19% more rapidly than low urease cells. This frequency is about 10³ greater than that of other biochemical variants previously isolated from XD cells. The high urease activity gradually declined in cells transferred from urea to other nitrogen sources, but rose rapidly when such cells were returned to urea, indicating the existence within the cells of some form of record of their ancestors' growth on urea. The data indicate the existence of a mechanism for generation, at unusually high frequency, of metastable variants with high urease activity. This mechanism, coupled with enrichment for the variants' progeny by virtue of their higher multiplication rate on urea, can account for the observed slow increase in urease activity of the population. It is suggested that the molecular basis of the urease increase may be gene amplification, based on animal cell models. An alternative hypothesis, namely a specific response induced in all cells by urea and manifested as a very slow adaptive increase in urease, has not been ruled out.     

Induction of urease by urea analogues in Evernia prunastri thallus

Urease activity in Evernia prunastri (L.) Ach. thallus is induced by incubation of lichen samples on 20 m M N,N-dimethylformamide and 20 m M N-formylurea or 40 m M thiourea although, in these two last cases, activity subsequently decreases again. The induction of enzyme activity is repressed by including 40 μ M cycloheximide in the medium. Filtration through Sepharose 6B of cell-free extracts from thalli incubated on 20 m M N,N-dimethylformamide shows a main peak of urease activity which has a molecular weight of about 560000 dalton. However, those extracts from thalli floated on 20 m M N-formylurea and 40 m M thiourea show several peaks of similar enzyme activity, which have molecular weights of about 1 100000, 670000, 260000 and 140000 dalton and 1 100000, 670000 and 140000 dalton respectively.
A time-course of urease activity could be related to the accumulation of lichen phenols in the thallus for samples incubated on N,N-dimethylformamide and thiourea.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

Shear deformation effects in enzyme catalysis. Metal ion effect in the shear inactivation of urease.

The mechanism of the inactivation of the enzyme urease produced by subjecting its dilute solutions to hydrodynamic shear stresses in the range 0.5-2.5 Pa has been determined. By studying the kinetics of urease-catalyzed urea hydrolysis during application of hydrodynamic shear under varying chemical environments, we demonstrate that micromolar quantities of metal ions, in this case adventitious Fe, can accelerate the oxidation of thiol groups on urease and thus inactivate it when the protein is subjected to a shearing stress of order 1.0 Pa. In the absence of metal ion this stress level is ineffectual. It is proposed that this type of synergy between deformation and chemical environment may be crucial in many situations where biological macromolecules are subjected to mechanical stress.     

Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations.