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Condensation products of urea and different aldehydes (formaldehyde, isobutyraldehyde, crotonaldehyde) are used in large amounts (more than 300,000 tons per year) as resins, binders, and insulating materials for industrial applications, as well as in controlled-release nitrogen fertilizer for greens, lawns, or bioremediation processes. The biodegradability of these condensates and the enzymic mechanism of their degradation was studied in mircoorganisms isolated from soil, which were able to use these compounds as the sole source of nitrogen for growth. Different pure cultures of both gram-positive and gram-negative bacteria completely degraded methylenediurea, dimethylenetriurea, isobutylidenediurea, and crotonylidenediurea to urea, ammonia, and the corresponding aldehydes and carbon dioxide. Enzymes initiating this degradation were purified and characterized and turned out to be different with regard to their regulation of expression, their physicobiochemical properties, and their reaction mechanism.

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.

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 γ-N-methylglutamine.

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.

The biodegradation of urea and condensation products thereof (ureaforms or methyleneureas), their nitrification, and their influence on the respiratory rate of soil was studied over periods of up to 100 days. The total methyleneurea content of the soil was determined after its acidic extraction, using a convenient colorimetric assay, and an HPLC protocol was established to analyze for specific components of methyleneureas. Urea, unfractionated methyleneureas, and hot-water soluble methyleneureas were rapidly metabolized to ammonium, which accumulated to high concentrations and was consequently oxidized to nitrate; an accumulation of nitrite was observed during urea but not during methyleneurea degradation. Hot water-insoluble methyleneureas were degraded much more slowly, and ammonium formed from these compounds was oxidized to nitrate without being released in significant amounts. These results suggest that the use of methyleneureas of optimized composition with regard to their water solubility may help to resolve problems such as the toxicity of ammonia to plant growth as well as nitrogen loss by leaching of nitrate, denitrification and volatilization.

An enzyme hydrolyzing the condensation products of urea and formaldehyde (ureaform) was purified and characterized from a bacterium isolated from soil and described as Ochrobactrum anthropi UF4. The enzyme designated as methylenediurea amidinohydrolase (methylenediurea deiminase) hydrolyzed ureaform condensation products of different length (methylenediurea, dimethylenetriurea, trimethylenetetraurea) to ammonium, formaldehyde, and urea at molar ratios of 2:1:1 (methylenediurea), 4:2:1 (dimethylenetriurea), and 6:3:1 (trimethylenetetraurea). Two other substrates, ureidoglycolate and allantoate, were also hydrolyzed, yielding glyoxylate and urea (ureidoglycolate) and glyoxylate, urea, and ammonium (allantoate), respectively. The molecular mass of the enzyme was determined by size exclusion chromatography to be 140 ± 25 kDa; the enzyme was composed of identical subunits of 38 ± 5 kDa, indicating that the native enzyme has a tetrameric structure. Growth of the bacterium in the presence of ureaform specifically induced the methylenediurea deiminase and no complete repression of enzyme synthesis by ammonium was observed.Key words: ureaformaldehyde, methylenediurea deiminase, fertilizer, Ochrobactrum anthropi.

Among several bioassays, the Scenedesmus subspicatus chlorophyll fluorescence test and the Photobacterium phosphoreum bioluminescence assay were selected to examine their applicability for toxicity evaluation of changes in coke plant effluent quality. In addition to the ecotoxicological parameters, a chemical analysis of dissolved organic carbon (DOC), ammonia-N, and nitrate-N was performed. It was demonstrated that the toxicity values obtained from the bioassays give a first indication on potential hazard and successful treatment. Decreasing toxicity values went along with decreasing DOC or ammonia-N values, but the sensitivity toward DOC reduction was higher. Sensitivity toward pure compounds was assessed by comparing the EC50 values of the luminescent bacteria assay with the corresponding EC50 values of the algal bioassay. The data showed a poor correlation between the two bioassays. © by John Wiley & Sons, Inc.

NAD-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.2) was purified to homogeneity from Sporosarcina ureae DSM 320; the native enzyme (Mr 250,000±25,000) is composed of subunits identical in molecular mass (Mr 42,000±3,000), suggesting a hexameric structure. In cell-free extracts and in its purified form, the enzyme was heat-stable, retaining 50% activity after 15 min incubation at temperatures up to 82°C. When exposed to low temperatures at pH values between 7.0 and 9.0. cell-free extracts and purified preparations lost enzyme activity rapidly and irreversibly. The addition of substrates, glycerol, or sodium chloride improved the stability of the enzyme with respect to cold lability and heat stability.

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.

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.

The Staphylococcus xylosus C2a urease was purified 655-fold to a maximum specific activity of 1 573 U/mg and contained three non-identical subunits (64, 17.8 and 16.3 kDa). The purified urease had a total molecular mass of 300 kDa and contained 3.91 ± 0.1 g atoms nickel per molecule as determined by atomic absorption spectrometry. The maximum urease activity occurred at pH 7.2 and 65°C, with a p I between 4 and 5.

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.

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.

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.

Summary: 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 K m 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.

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.

Summary The number of thiobacilli and the thiosulfate oxidation potency were determined in the water and in the sediment of the river Saar in order to study the aerobic part of the sulfur cycle. Samples were taken along the entire course of the river between summer 1978 and spring 1981, i.e. before the lower part of the river was canalized. In the water samples, a close correlation was observed between the total number of thiobacilli and the thiosulfate oxidation potency. In the sediment of the middle part of the river, which is canalized, the observed concentration and metabolic activity of the thiobacilli, the presence of sulfide and the diminished concentration of sulfate indicated that both oxidation and reduction of sulfur occurred in this reach. Highest concentrations (numbers) and activities of thiobacilli, both in the water and in the sediment, were observed at low waters in August 1978; 10 3 cells/ml water and 10 5 cells/g sediment were detected. Lowest concentrations and activities were measured during high waters in February 1980. In a longitudinal study of the river the following species were enumerated in water and sediment by means of selective media: T. thioparus, T. neapolitanus, T. denitrificans, T. thiooxidans and T. ferrooxidans . Downstream from the source, the number of all these species increased, but in case of T. thioparus and T. neapolitanus maximum concentrations were already observed in the upper part of the river, which is not canalized and not polluted by industrial wastes. The remaining three species increased in number along the entire river. The concentrations of the individual species were considerably higher in the sediments than in the overlaying water. In general, the detected numbers were about 10 3 -times higher in the sediment samples, regardless whether selective or non-selective media were used.

With 0.5% substrate present in mineral medium, cells of Alcaligenes eutrophus H 16 were able to grow heterotrophically at the expense of guanine, hypoxanthine and xanthine, but not of adenine as sole sources of carbon and nitrogen. An increase in cell counts, however, was observed at lower adenine concentrations (0.1%). Similarly, adenine was only respired if present at low concentrations. Higher amounts of adenine were inhibitory to the utilization of adenine, guanine, hypoxanthine, xanthine, allantoin and glyoxylate, but not to that of fructose or glycerate. The adenine-dependent inhibition of adenine utilization was not overcome by the addition of thiamine, uridine or cytidine. The enzyme glyoxylate carboligase, usually formed in presence of metabolisable purines and of allantoin, was synthesized only at low adenine concentrations. Higher amounts were inhibitory even with allantoin present as additional substrate. According to these resutls, the utilization of purine derivatives and of allantoin as sources of carbon and energy is repressed by adenine in cells of A. eutrophus H 16.

The uptake of adenine, guanine, xanthine, hypoxanthine and uric acid by whole cells was studied, using spectrophotometric techniques, 14C-labelled compounds and metabolic inhibitors. Three different non-constitutive systems were shown to maintain the uptake of adenine and that of the pairs guanine/hypoxanthine and xanthine/uric acid. —Active transport of adenine was induced by adenine only, but passive uptake was also involved. Maximum K T values of 110–131 μM were observed at the pH optimum of 8.0. —Guanine and hypoxanthine were translocated by one single mechanism as indicated by K T and K I values. This system was induced by both these substances but its affinity was 51/2-times higher for guanine than for hypoxanthine; it was noncompetitively stimulated by Mg2+. — A further system, induced by xanthine and uric acid, catalyzed the uptake of both these compounds. It exhibited two pH optima (at pH 6.6 and 7.9); inactivation by heat and stimulation or inhibition by several compounds indicated that two separate mechanisms might be involved in the uptake of xanthine and uric acid.

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.

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 K m-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.

The uptake of the radioactive ammoniumanalogue 14C-methylammonium1 was measured in heterotrophically grown cells of Alcaligenes eutrophus H16 in order to study the mechanism of NH 4 + uptake. MA gradients of up to 200 were built up by a substrate-specific and energy-dependent system which showed a K m of 35–111 μM and a V max of 0.4–1.8 nmol MA/min per mg protein. The involved carrier exhibited a higher affinity towards NH 4 + than towards CH3NH 3 + indicating that ammonium rather than MA was its natural substrate. Cold shock with hypotonic but not with hypertonic solutions caused the efflux of almost the entire accumulated MA. Osmotic shock did not affect the uptake reaction, suggesting that no periplasmic binding proteins were involved. Indirect observations indicate the membrane potential as driving force for MA uptake. High rates of uptake were observed in cells grown under nitrogen deficiency or with nitrate as nitrogen source. The uptake rate decreased during growth at high ammonium concentrations indicating that biosynthesis of nitrogenous compounds was supported by passive diffusion of NH3. The data suggest that the formation of the carrier is subject to “nitrogen control”.

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.

BeiBacillus fastidiosus, der Harnsaure und Allantoin uber Glyoxylsaure abbaut, wurde der Glyoxylat-Stoffwechsel untersucht. Enzyme des Glycin-Serin-Weges, des Oxalat-Weges und des β-Hydroxyaspartat-Weges waren in zellfreien Extrakten nicht nachweisbar. Das Enzym Glyoxylatcaboligase, welches die Synthese von Tartronsauresemialdehyd (TSA) aus Glyoxylsaure katalysiert, war zwar in den Extrakten vorhanden,abereine nachfolgende Umsetzung von TSA uber den Glycerat-Weg schien unwahrscheinlich, da keine Glyceratkinase nachgewiesen werden konnte. Allerdingswurde eine enzymatische Tautomerisierung von Enzymen, welche die Synthese von Pyruvat aus HP uber Serin katalysieren,deutet darauf hin, das die beobachtete enzymatische Umwandlung von TSA zu HP in diesem Organismus an der Synthese von C3-Verbindungen aus Glyoxylat beteiligt ist.

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.

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.

Die Kinetik der Aufnahme von 63Ni wurde an zwei Stammen von Alcaligenes eutrophus untersucht, die Nickelionen fur das chemolithotrophe Wachstum benotigen. Mit Kohlendioxid als einziger Kohlenstoffquelle wird das Wachstum durch niedrige Konzentrationen von Nickel gefordert, wobei das Optimum der Wachstumsforderung bei 0,3 μM Nickel lag. Hohere Nickelkonzentrationen wirkten hemmend. Das heterotrophe Wachstum mit Fructose wurde durch Nickelionen nicht gefordert. — Ubertragen 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 angehauft. Die beobachtete Ni-Aufnahme zeigte ein Temperaturoptimum bei etwa 29° C und wurde durch Hemmstoffe wie Arsenit, Jodacetat, Methylenblau, Natriumazid und Natriumcyanid stark beeintrachtigt. Andere Schwermetallionen (Zn, Co, Mn und Cu) verminderten die Nickelaufnahme nur geringfugig. Durch 58NiCl2 und Toluol wurde der Efflux von 63Ni aus den Zellen gefordert. Die Beobachtungen lassen darauf schliesen, das Nickelionen durch einen energieabhangigen Prozes in chemolithotroph gewachsenen Zellen dieser Stamme angehauft werden.

Summary 8 mutants of Hydrogenomonas eutropha H 16 were isolated after treatment with the mutagen HNO 2 and enrichment in a medium containing urea and colistin. The mutants could no longer utilize urea and needed for optimal growth an increased concentration of NH 4 Cl which strongly inhibited the growth of the wild type. The more intensively investigated mutant U 5 showed an exponential growth with glutamate, but not with NH 4 Cl Like the wild type, the mutant also produced urease and NADP specific glutamate dehydrogenase; during nitrogen starvation an increase in specific urease activity was observed in both strains. The possibility of damage to the urea and ammonia transport system is discussed.

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.

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.

Anaerobically adapted and illuminated Scenedesmus evolves molecular hydrogen from endogenous organic compounds. This photoproduction of H2 does not require photosystem II, since 5x10(-6) M DCMU, which inhibited normal photosynthesis almost completely, did not significantly inhibit the photoevolution of H2. The relative efficiencies in far-red light of photosynthesis, photoreduction and H2 production were determined. Photohydrogen evolution was comparatively the most efficient of these three processes. Three mutants of Scenedesmus (isolated and characterized by Dr. N. I. Bishop) were also tested. Mutant PS-50, which lacks cytochrome 552, did not photoproduce H2. Mutant No. 11, blocked in photosystem II, showed rates of H2 production comparable to those of the wild type. Cl-CCP, an uncoupler of photophosphorylation, caused an apparent stimulation of H2 production by mutant No. 11 and wild-type cells. Mutant No. 8, which is partially blocked in photosystem I, showed a diminished photohydrogen production which was inhibited by Cl-CCP. These results suggest that photoproduction of hydrogen by photosystem I is due either to cyclic photophosphorylation, which supplies energy needed for a dark, H2-yielding reaction, or to a more direct photooxidation of organic compounds by the photosynthetic electron transfer chain.