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H Kaltwasser

Publications and source records attributed to H Kaltwasser.

At least 19 recordsLinked to original sources
PubMed2000

Ammonium and methylammonium uptake in a fertilizer-degrading strain of Ochrobactrum anthropi.

The transport of ammonium and methylammonium was studied in a strain of Ochrobactrum anthropi, a microorganism isolated from garden soil and able to degrade methyleneureas which are used as slow-release nitrogen fertilizer. The activity of both transport systems was determined using [14C]methylammonium. Differences between the two transport systems were observed with regard to their pH- and temperature dependence as well as their kinetic parameters and regulation during growth with various nitrogen sources. Ammonium transport was subject to repression by ammonium and to derepression in its absence, while the methylammonium carrier was induced in the presence of methylamine. The ammonium but not the methylammonium transport system was severely inhibited by ammonium, and metabolic poisons inhibited both uptake systems. The analysis of intracellular metabolites using thin-layer chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that methylammonium was rapidly metabolized to N-methylglutamate via gamma-N-methylglutamine.

Biodegradation, Environmental↗
PubMed1998

Microbial urea-formaldehyde degradation involves a new enzyme, methylenediurease.

The enzymic mechanism of metabolization of urea-formaldehyde condensation products (methyleneureas; MU) and the fate of the degradation products ammonium, urea and formaldehyde were studied in bacteria isolated from garden soil, which were able to use methyleneureas as the sole source of nitrogen for growth. An organism identified as Ochrobactrum anthropi completely degraded methylenediurea (MDU) and dimethylenetriurea (DMTU) to urea, ammonia, formaldehyde and carbon dioxide. An enzyme designated as methylenediurease (methylenediurea deiminase; MDUase) was responsible for the degradation of both MDU and DMTU as well as higher polymerized MU. Growth on MU as the nitrogen source specifically induced the synthesis of this enzyme, which seems to be located in the periplasm of the bacterium. Under these growth conditions, urease as well as NAD-specific formaldehyde and formiate dehydrogenase were expressed to high levels, efficiently using the products of MU degradation, and high-affinity transport systems for urea and ammonia were synthesized scavenging the environment for these products.

Amidohydrolases↗
PubMed1994

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

DNA sequence analysis of the structural urease genes from Staphylococcus xylosus revealed that three enzyme subunits are encoded in the order of 11,000, 15,400 and 61,000 (mol. mass), which correspond to the single polypeptide chain of jack bean urease (90,800). 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 (alpha beta gamma)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 indicate that in these staphylococcal strains urea hydrolysis might function in a manner similar to the peptide bond cleavage by chymotrypsin.

Amino Acid Sequence↗
PubMed1994

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 420,000; it consisted of subunits with molecular weights of 72,400 (alpha), 20,400 (beta), 13,900 (gamma) in an estimated (alpha beta gamma)4 stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420,000 to 100,000. 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.

Amino Acid Sequence↗
PubMed1993

Cloning, heterologous expression, and sequencing of the Proteus vulgaris glnAntrBC operon and implications of nitrogen control on heterologous urease expression.

The glnAntrBC operon of Proteus vulgaris was cloned and heterologously expressed in Escherichia coli. The nucleotide sequence was determined. An open reading frame of 1407 bp was identified as the glnA gene and the deduced amino acid sequence showed 82% identity with the E. coli glutamine synthetase protein. Heterologous expression of the glnA gene in E. coli restored glutamine synthetase (GS) activity in a GS-negative mutant and a 52 kDa protein was detected and addressed as the GS subunit of P. vulgaris. Adjacent to the glnA gene the regulatory genes ntrB and ntrC were identified. Their coding regions comprised 1053 and 1452 bp, respectively, and the deduced gene products NRII (NtrB) and NRI (NtrC) shared 72% identity with the corresponding E. coli proteins. Heterologous expression in E. coli revealed only a 54 kDa protein which was shown to be NRI. NRII was not detectable using the methods employed.

Amino Acid Sequence↗
PubMed1993

Properties of the cold-labile NAD(+)-specific glutamate dehydrogenase from Bacillus cereus DSM 31.

Nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Bacillus cereus DSM 31 was enriched 260-fold. The molecular mass was determined by gel filtration to be 270 kDa (+/- 25 kDa). The enzyme was highly specific for the coenzyme NAD(H) and catalysed both the formation and the oxidation of glutamate. Apparent Km values of 7.7 mM for glutamate and 0.56 mM for NAD+ during oxidative deamination were measured. Both in crude cell-free extracts and in enriched preparations the enzyme was extremely unstable, especially at low temperatures. The loss of activity in the cold was found to be due to the dissociation of the holoenzyme into catalytically inactive subunits of molecular mass 48 kDa (+/- 5 kDa), indicating that the native enzyme has a hexameric structure. The activity was restored under certain conditions, and no instability of the enzyme in the cold was observed in undisrupted cells.

Bacillus cereus↗
PubMed1992

Creatinine and N-methylhydantoin degradation in two newly isolated Clostridium species.

With N-methylhydantoin (NMH) as the main organic substrate, two strictly anaerobic spore forming Gram-positive bacterial strains were isolated from sewage sludge. These strains, named Clostridium sp. FS23 and Clostridium sp. FS41, totally degraded NMH, via N-carbamoylsarcosine (CS) and sarcosine as intermediates. Strain FS23 grew also with creatinine, which was converted to NMH by creatinine iminohydrolase (EC 3.5.4.21). This enzyme was formed at high rates with all substrates tested. Cytosine and 5-fluorocytosine were not utilized as substrates by creatinine iminohydrolase preparations purified to a homogeneity of 98%. NMH amidohydrolase (NMHase) and N-carbamoylsarcosine amidohydrolase (CSHase) turned out to be inducible in both strains. Other than in aerobic organisms, NMHase from these two isolated did not require ATP for enzymatic activity. SH-group protecting agents were not necessary for stability.

Adenosine Triphosphate↗
PubMed1991

Cloning and expression of various staphylococcal genes encoding urease in Staphylococcus carnosus.

The urease genes from Staphylococcus xylosus C2a, Staphylococcus aureus U500, and S. aureus Newman were cloned in Staphylococcus carnosus using the plasmid vectors pCA43 and pCA44. The resulting respective recombinant plasmids pUra 402, pUraUH66, and pUra17 contained chromosomal DNA fragments with sizes of 5.6, 5.8, and 6.8 kb, respectively. Investigations on urease expression of the donor and recombinant strains in media with various nitrogen sources revealed that S. xylosus C2a produced urease constitutively at the highest specific activity. All of the recombinant strains had significantly lower urease activities than their DNA-donor strains. The nickel-dependence of urease was demonstrated in S. aureus U500 by a plate diffusion assay.

Cloning, Molecular↗
PubMed1990

Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes.

A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease.

Amino Acid Sequence↗
PubMed1990

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 unavailable for maximal expression of enzymatically active urease; this inhibition of urease 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 performed apo-urease.

Amino Acids↗
PubMed1990

Uptake and metabolism of methylammonium by Pseudomonas aeruginosa.

The mechanism of ammonium uptake was studied in Pseudomonas aeruginosa, measuring the uptake (transport and metabolism) of [14C]methylammonium (MA). This ammonium analogue was not utilized for growth, but unmetabolized MA was accumulated to intracellular concentrations about 30 times higher than those in the medium. Most of the MA taken up, however, was rapidly metabolized to gamma-N-methylglutamine, which could be removed from the cells by the addition of ammonium. Uptake of MA exhibited distinct optima at pH 7.0 and 35 to 40 degrees C and depended on metabolic energy, as indicated by the inhibitory effect of various metabolic poisons. Growth with ammonium as nitrogen source resulted in the repression of MA uptake, whereas high uptake rates were observed with nitrate or after incubation without nitrogen source. These results suggested that the ammonium/MA uptake system is subject to nitrogen control in P. aeruginosa.

Azides↗
PubMed1989

Energy-dependent uptake of urea by Bacillus megaterium.

Evidence for the existence of an energy-dependent urea uptake system in Bacillus megaterium DSM 90 was obtained by studying the uptake of 14C-urea. In vivo urea uptake and in vitro urease activity differed significantly with respect to temperature- and pH-dependence, kinetic parameters and response towards metabolic inhibitors. Highest uptake activities were observed during exponential growth, and a rapid decrease in urea uptake occurred when cells entered the stationary growth phase and started to sporulate. Significant differences in the uptake rates were observed during growth with different nitrogen sources, suggesting that the formation of the system is under nitrogen control.

Bacillus megaterium↗
PubMed1989

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, respectively. 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.

Alcaligenes↗
PubMed1986

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 mumol urea min-1 mg-1 at 30 degrees 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 230,000 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.

Bacillus↗
PubMed1982

Regulation by repression of urease biosynthesis in Proteus rettgeri.

Measuring the specific enzyme activity in cells of Proteus rettgeri it was shown that urease formation is controlled by repression through ammonia. Derepressed synthesis of the enzyme, as initiated by the absence of ammonia, required an external nitrogen source, which may not only be urea, but also nitrate, glutamate or nutrient broth. In contradiction to earlier reports the observations indicated that urea is not required for the synthesis of this enzyme, and that, therefore, urease is not an inducible enzyme in this microorganism.

Culture Media↗
PubMed1982

Microbial methane oxidation in the River Saar.

In the River Saar, the distribution of methane-oxidizing bacteria and their metabolic activity were determined in vertical and longitudinal profiles. At the sediment surface about two orders of magnitude more methane oxidizers were detected than in the overlying water. In the river as well as in laboratory experiments, the rate of methane oxidation was closely related to the concentration of methane. Most of the methane produced by methanogenic bacteria in the the sediment escaped into the atmosphere. On the average only 1.2% of the produced methane was oxidized in the water phase.

Fresh Water↗
PubMed1977

[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.

Alcaligenes↗