Heinrich Kaltwasser

Evidence for carrier-mediated, energy-dependent uptake of urea in some bacteria

Evidence for the existence of an energy-dependent urea permease was found for Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5a1 by studying uptake of 14C-urea. Since intracellular urea was metabolized immediately, uptake did not result in formation of an urea pool. Evidence is based on observations that the in vivo urea uptake and in vitro urease activity differ significantly with respect to kinetic parameters, temperature optimum, pH optimum, response towards inhibitors and regulation. The Km for urea uptake was 15–20 times lower (38 μM and 13 μM urea for A. eutrophus and K. pneumoniae, respectively) than the Km of urease for urea (650 μM and 280 μM urea), the activity optimum for A. eutrophus was at pH 6.0 and 35°C for the uptake and pH 9.0 and 65°C for urease. Uptake but not urease activity in both organisms strongly decreased upon addition of inhibitors of energy metabolism, while in K. pneumoniae, potent inhibitors of urease (thiourea and hydroxyurea) did not affect the uptake process. Significant differences in the uptake rates were observed during growth with different nitrogen sources (ammonia, nitrate, urea) or in the absence of a nitrogen source; this suggested that a carrier is involved which is subject to nitrogen control. Some evidence for the presence of an energy-dependent uptake of urea was also obtained in Pseudomonas aeruginosa DSM 50071 and Providencia rettgeri DSM 1131, but not in Proteus vulgaris DSM 30118 and Bacillus pasteurii DSM 33.

Ammonium assimilation in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae

No active uptake of ammonium was detected in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae, which indicates that these bacteria depend on the passive diffusion of ammonia across the cell membrane. In P. vulgaris the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and glutamate dehydrogenase (GDH) were present, and these enzymes exhibited high affinities for ammonium. In B. pasteurii and S. ureae, however, no GS activity was detected, and GOGAT activity was only present in S. ureae. GDH enzymes were present in these two organisms, but showed only low affinity for ammonium, with apparent Km-values of 55.2 mM in B. pasteurii and 36.7 mM in S. ureae, repectively. These observations explain why P. vulgaris is able to grow at neutral pH and low ammonium concentration (2 mM), while B. pasteurii and S. ureae require high ammonium concentration (40 mM) and alkaline pH for growth.

Nickel-content of urease from Bacillus pasteurii.

Urease from Bacillus pasteurii DSM 33 was purified 34-fold to a maximum specific activity of 996.5 μmol urea min-1 mg-1 at 30°C. Homogeneity was demonstrated by isoelectric focussing which showed a single protein zone corresponding to a pI of about 4.6. The native enzyme was demonstrated to have a molecular mass of 230000 and to consist of identical subunits of 65 500, as measured by SDS electrophoresis. Radioactive 63Ni-nickel co-chromatographed with urease through gel filtration, ion-exchange, and affinity chromatography. Measuring specific radioactivity, the nickel content was found to be 1.00 (±0.1) g-atom Ni per mol of subunit, and 0.82 g-atom Ni per mol as measured by atomic absorption spectrometry. This indicates that 1 atom of nickel is present in each of four subunits of the enzyme.

Urease from Arthrobacter oxydans, a nickel-containing enzyme

In Arthrobacter oxydans, Klebsiella aerogenes and Sporosarcina ureae, growth with urea as a nitrogen source turned out to be more sensitive to inhibition by EDTA than that with ammonia. The inhibition was overcome by added nickel chloride, but not by other divalent metal ions tested. In A. oxydans the uptake of 63Ni was paralleled by an increase in urease (urea amidohydrolase, EC 3.5.1.5) activity under certain conditions. Following growth with radioactive nickel, urease from this strain was enriched by heat treatment and acetone fractionation. Copurification of 63Ni and urease was observed during subsequent Sephadex gel chromatography. Almost the entire labelling was detected together with the purified enzyme after focusing on polyacrylamide gel. The relative molecular mass of the purified urease was estimated to be 242,000. The pH optimum was 7.6, the Km-value 12.5 mmol/l and the temperature optimum 40°C; heat stability was observed up to 65°C. In presence of 10 mmol/l EDTA the protein-nickel binding remained intact at pH 7; at pH 5 and below, nickel was irreversibly removed with concommitant loss of enzyme activity. The results demonstrated that nickel ions are required for active urease formation in the bacterial strains studied, and that urease from A. oxydans is a nickel-containing enzyme.

Nickel requirement for chemolithotrophic growth in hydrogen-oxidizing bacteria

In a comparative study the requirement of several strains of autotrophic hydrogen-oxidizing bacteria for nickel was examined. Autotrophic growth was studied both in liquid media, previously freed from trace metals; and on solidified media, using a plate diffusion assay. The latter assay was based on the observation that EDTA causes complete inhibition of autotrophic growth on agar medium as a result of nickel deficiency. Nickel was shown to be required as a trace element in five strains of Alcaligenes eutrophus, in two strains of Xanthobacter autotrophicus, in Pseudomonas flava, in Arthrobacter spec. 11X and in strain 12X. In these bacteria nickel was not replaceable by cobalt, copper, manganese or zinc ions. No significant nickel requirement was detected by these methods, however, for Paracoccus denitrificans and Nocardia opaca 1b.

Isolation of a novel, phosphate-activated glutaminase from Bacillus pasteurii

In Bacillus pasteurii glutamine is being taken up efficiently by a sodium-dependent uptake system and subsequently hydrolysed to ammonium and glutamate. Concerning the latter process, a catabolic L-glutamine amidohydrolase (glutaminase) was isolated from the cytoplasm of this alkaliphilic bacterium and purified to homogeneity using liquid chromatography. Biochemical and physical parameters of the pure enzyme were examined in detail. Interestingly, analysis of the glutaminase revealed a marked increase in catalytic activity in the presence of phosphate, a property yet restricted to animal glutaminases. This is the first report on the presence of a phosphate-activated glutaminase in bacteria.

[Energy-dependent 63Ni-uptake by Alcaligenes eutrophus strains H1 and H16 (author's transl)].

Kinetic studies of the uptake of 63Ni were undertaken with two strains of Alcaligenes eutrophus, known to require nickel ions for chemolithotrophic growth. Using carbon dioxide as sole carbon source, growth is stimulated by low concentrations of nickel with optimum concentration for growth stimulation at about 0.3 micron nickel. Higher nickel concentrations were inhibitory. Heterotrophic growth on fructose was not stimulated by nickel ions.--Upon transfer into phosphate buffer freed of heavy metal ions, autotrophically grown cells exhibited rapid uptake of 63Ni which was dependent upon the presence of hydrogen, oxygen and carbon dioxide. Within 60 min nickel was accumulated from the medium, reaching 280-fold concentration in the cells. The observed uptake exhibited a temperature optimum at about 29 degrees C and was markedly inhibited by metabolic inhibitors such as arsenite, iodoacetate, methylene-blue, sodium azide and sodium cyanide. Other heavy metal ions (Zn, Co, Mn and Cu) only slightly inhibited 63Ni-uptake. The efflux of 63Ni from the cells was stimulated by 58NiCl2 and by toluene. These data indicate that nickel ions are accumulated by an energy dependent mechanism in chemolithotrophically grown cells of these strains.Kinetic studies of the uptake of 63Ni were undertaken with two strains of Alcaligenes eutrophus, known to require nickel ions for chemolithotrophic growth. Using carbon dioxide as sole carbon source, growth is stimulated by low concentrations of nickel with optimum concentration for growth stimulation at about 0.3 μM nickel. Higher nickel concentrations were inhibitory. Heterotrophic growth on fructose was not stimulated by nickel ions.-Upon transfer into phosphate buffer freed of heavy metal ions, autotrophically grown cells exhibited rapid uptake of 63Ni which was dependent upon th presence of hydrogen, oxygen and carbon dioxide. Within 60 min nickel was accumulated from the medium, reaching 280-fold concentration in the cells. The observed uptake exhibited a temperature optimum at about 29° C and was markedly inhibited by metabolic inhibitors such as arsenite, iodoacetate, methylene-blue, sodium azide and sodium cyanide. Other heavy metal ions (Zn, Co, Mn and Cu) only slightly inhibited 63Ni-uptake. The efflux of 63Ni from the cells was stimulated by 58NiCl2 and by toluene. These data indicate that nickel ions are accumulated by an energy dependent mechanism in chemolithotrophically grown cells of these strains.ZusammenfassungDie Kinetik der Aufnahme von 63Ni wurde an zwei Stämmen von Alcaligenes eutrophus untersucht, die Nickelionen für das chemolithotrophe Wachstum benötigen. Mit Kohlendioxid als einziger Kohlenstoffquelle wird das Wachstum durch niedrige Konzentrationen von Nickel gefördert, wobei das Optimum der Wachstumsförderung bei 0,3 μM Nickel lag. Höhere Nickelkonzentrationen wirkten hemmend. Das heterotrophe Wachstum mit Fructose wurde durch Nickelionen nicht gefördert. — Übertragen in Phosphatpuffer, der von Schwermetallionen befreit worden war, zeigten autotroph gewachsene Zellen eine rasche Aufnahme von 63Ni, sofern Wasserstoff, Sauerstoff und Kohlendioxid zugegen waren. Dabei wurde Nickel innerhalb von 60 min aus dem umgebenden Medium bis zur 280 fachen Konzentration in den Zellen angehäuft. Die beobachtete Ni-Aufnahme zeigte ein Temperaturoptimum bei etwa 29° C und wurde durch Hemmstoffe wie Arsenit, Jodacetat, Methylenblau, Natriumazid und Natriumcyanid stark beeinträchtigt. Andere Schwermetallionen (Zn, Co, Mn und Cu) verminderten die Nickelaufnahme nur geringfügig. Durch 58NiCl2 und Toluol wurde der Efflux von 63Ni aus den Zellen gefördert. Die Beobachtungen lassen darauf schließen, daß Nickelionen durch einen energieabhängigen Prozeß in chemolithotroph gewachsenen Zellen dieser Stämme angehäuft werden.

Urease from Staphylococcus saprophyticus: purification, characterization and comparison to Staphylococcus xylosus urease

Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420000; it consisted of subunits with molecular weights of 72400 (α), 20400 (β), and 13900 (γ) in an estimated (αβγ)4 stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420000 to 100000. In the native enzyme, 4.09 (±0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.

Mechanism of Microbial Degradation of Slow-Release Fertilizers

Methyleneureas are condensation products of urea and formaldehyde of different molecular mass and solubility; they are used in large amounts both as resins, binders, and insulating materials for industrial applications, as well as a slow-release nitrogen fertilizer for greens, lawns, or in bioremediation processes. In the present study, the microbial breakdown of these products was investigated. The nitrogen was released as ammonia and urea, and the formaldehyde released immediately oxidized via formiate to carbon dioxide. The enzymatic mechanism of metabolization of methyleneureas was studied in microorganisms isolated from soil, which were able to use these compounds as the sole source of nitrogen for growth. A strain of the Gram-negative bacterium Ralstonia paucula (formerly Alcaligenes sp. CDC group IVc-2) completely degraded methylenediurea and dimethylenetriurea to urea, ammonia, formaldehyde, and carbon dioxide. The enzyme initiating this degradation (methylenediurease) was purified and turned out to be different from the previously described enzyme from Ochrobactrum anthropi with regard to its regulation of expression and physicobiochemical properties. Fungal degradation of methyleneureas may occur via the formation of organic acids, thus leading to a nonenzymatic degradation of methyleneureas, which are unstable under acidic conditions.

Threonine is present instead of cysteine at the active site of urease from Staphylococcus xylosus.

DNA sequence analysis of the stuctural urease genes from Staphylococcus xylosus revealed that three enzyme subunits are encoded in the order of 11000, 15400 and 61000 (mol. mass), which correspond to the single polypeptide chain of jack bean urease (90800). Comparing the deduced amino acid sequence of S. xylosus urease with the amino acid sequence of jack bean urease an overall portion of 56% identical residues was found. For S. xylosus urease a subunit structure of (αβγ)4 was proposed, based on the comparison of the deduced amino acid content of the enzyme subunits with the total amino acid content of the purified enzyme. The staphylococcal enzyme contained no cysteine, as deduced from DNA sequence and confirmed by the determination of the total amino acid content in the purified enzyme. Instead of cysteine, known to be catalytically essential in the plant enzyme, and conserved among all bacterial ureases analyzed so far, threonine was found in S. xylosus. This amino acid-exchange was located within a highly conserved domain of 17 amino acids, supposed to be part of the active site. Sequence analysis of the respective region of Staphylococcus saprophyticus urease showed that it also contains threonine instead of cysteine. In contrast to jack bean urease S. xylosus urease was not affected by the SH-group inhibitor dipyridyl disulfide but was completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The presented results indicated that in these staphylococcal strains urea hydrolysis might function in a manner similar to the peptide bond cleavage by chymotrypsin.