Diethelm Kleiner

Arginine ammonification, a simple method to estimate microbial activity potentials in soils

Abstract A simple, rapid and inexpensive method to determine microbial activity potentials, based on ammonification of arginine, was developed and tested on bacterial cultures and soil samples. The results are highly reproducible and correlate well with respiratory activities. Ammonification starts immediately after the addition of arginine and is linear for more than l h. Both properties show that physiological status and number of microorganisms remain stable during the assay.

A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane.

Aims:  To isolate and identify endophytic nitrogen‐fixing bacteria in sugarcane growing in Cuba without chemical fertilizers.

Whole cell respiration and nitrogenase activities in Azotobacter vinelandii growing in oxygen controlled continuous culture

Azotobacter vinelandii strain OP was grown in continuous culture at various dissolved oxygen concentrations of air (100% air saturation of the medium=225 ±14 μM O2). Sucrose was added as carbon source and either dinitrogen or ammonia as nitrogen sources. Irrespective of the nitrogen source steady state cultures showed the following general responses with dissolved oxygen concentrations increasing from about 1% to 30% air saturation: (i) cell protein levels, (ii) the amount of cell protein formed per sucrose consumed as well as (iii) nitrogenase activity decreased by at least a factor of two while (iv) cellular respiration increased. At higher oxygen concentrations the parameters changed only slightly, if at all. Increasing the sucrose concentration in the inflowing medium (sR) from 3 g/l to 15 g/l increased the total level of cellular respiration with nitrogen-fixing cultures but was more pronounced with ammonium-assimilating cultures. With nitrogen-fixing cultures cell protein levels increased five-fold while the ratio of protein formed per sucrose consumed as well as cellular nitrogenase activity remained unaffected. With ammonium-assimilating cultures the cell protein level was only doubled and the level of cell protein formed per sucrose consumed was decreased at the higher sR.Increasing the dilution rate at a constant oxygen concentration of 45% air saturation resulted in an almost parallel increase of both cellular respiratory and nitrogenase activity at low and moderate dilution rates. At high dilution rates nitrogenase activity increased steeply over the respiratory activity. Nitrogen-fixing cultures adapted to various oxygen concentrations were subjected to oxygen stress by increasing the oxygen concentration for 7 min. In all cases, this resulted in a complete inhibition (‘switch-off’) of nitrogenase activity. Upon restoration of the original oxygen concentration nitrogenase activity returned to a decreased level. The discussion arrives at the conclusion that some of the results are incompatible with the concept of respiratory protection of nitrogenase.

Energy expenditure for cyclic retention of NH3/NH4+ during N2 fixation by Klebsiella pneumoniae

The permeability coefficeint for NH3 through bacterial membranes was determined to be around 2 × 10−3 cm·s−1. This value was used to calculate the outward diffusion of NH3 from intracellular pools and the energy costs for reabsorption as NH4 +. For an N2‐fixing continuous culture of Klebsiella pneumoniae an energy expenditure of around 4 ATP per NH3 produced was calculated, thus increasing significantly the energy requirement for N2 fixation in vivo.

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.

Inhibition of ammonium (methylammonium) transport in Klebsiella pneumoniae by glutamine and glutamine analogues

Ammonium-transport systems are widely distributed among bacteria [1-8]. In several strains the synthesis of these transport systems is regulated by the nitrogen source [5-7]. Nothing is known, however, about the regulation of the activity of the transport systems. By employing the glutamine analogues methionine sulfone (MSF), methionine sulfoximine (MSX) and 6-diazo-5-oxo-norleucine (DON), which are well known as inhibitors of the enzymes glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.1.13), we found evidence for a regulation of ammonium transport by the intracellular glutamine level.

Some properties of a Klebsiella pneumoniae ammonium transport negative mutant (Amt

The main property of an Amt- (ammonium transport negative) mutant of Klebsiella pneumoniae is its inability to accumulate NH4+intracellularly. When growing on nitrogen sources other than NH4+, the mutant constantly looses NH3 by diffusion. This loss results in poor growth. The NH3 excretion suggests the existence of a futile cycle (NH3 loss/NH4+reabsorption) in the wild type and possibly other bacterial strains, which do not constantly excrete NH3.

Regulatory aspects of inorganic nitrogen metabolism in the Rhodospirillaceae

Regulatory aspects of the assimilation of inorganic nitrogen compounds (ammonia, nitrate, nitrogen) were studied in 12 strains belonging to the Rhodospirillaceae. All strains possessed an ammonium transport system, as demonstrated by 14C-methylammonium uptake. This uptake showed saturation kinetics (Km between 50–150 μM), and was competitively inhibited by ammonium (Ki between 5–18 μM). The ammonium transport systems were repressed by ammonium in the growth medium. The nitrogenase activity of all strains was reversibly inhibited by ammonium (“switch-off”). This effect was not shown under nitrogen starvation conditions with the exception of some strains of Rhodopseudomonas capsulata, the nitrogenase of which was always susceptible to switch-off by ammonium. Assimilation of nitrate was confined to some strains of Rhodopseudomonas capsulata.

Evidence for an ammonium transport system in the N2-fixing phototrophic bacteriumRhodospirillum rubrum

An ammonium transport system in the phototrophic N2-fixing bacteriumRhodospirillum rubrum was characterized by using the uptake of14C-methylamine as a probe.Uptake showed saturation kinetics with an apparentKm=110 μM. It was competitively inhibited by ammonium (Ki=7 μM). Uptake exhibited a narrow pH maximum around pH 7.0.Up to 200-fold gradients across the membrane were formed within 40–60 min. Gradient formation was inhibited by carbon starvation, azide or cyanide. Pre-accumulated methylamine was released by ammonium pulses to more than 80%, indicating only minor metabolization.The synthesis of the transport system was repressed by ammonium in high concentrations.

Polyhalogenated benzo- and naphthoquinones are potent inhibitors of plant and bacterial ureases

Polyhalogenated benzo‐ and naphthoquinones were found to be potent inhibitors of pure ureases from Bacillus pasteurii and Canavalia ensiformis. They also inhibited ureases in whole cells of Helicobacter pylori, Klebsiella oxytoca and Proteus mirabilis. Inhibition was non‐competitive with K i values in the micromolar range or below. Inhibition was irreversible as shown by equilibrium dialysis. Inhibitory power decreased considerably when halogens were replaced by –OH, –CN, alkoxy or alkyl groups.