Minimal size of Nitrobacter membrane frangments retaining nitrite oxidizing activity

Summary The minimal size of active membrane fragments from sonic homogenates of a Nitrobacter cell suspension is about 22 m. Nitrite oxidizing activity is absent from particles smaller than that although the distribution of cytochrome a, a link in the respiratory chain responsible for nitrite oxidation, does not show any abnormality below the critical size indicated.Two independent estimates of the parameters of the cumulative distribution function for particle size in sonic homogenates have been made using either statistics from electron micrographs or sedimentation pattern observed after density gradient centrifugation. The distribution function was found to be of the log-normal type which was considered to be a consequence of the observed kinetics of sonic comminution. Another estimate of the parameters of the distribution function was made by fixed angle centrifugation, a method much less accurate with respect to size determination but which allowed a rapid estimate of nitrite oxidizing capacity remaining in particles smaller than the given size.Results from the first two determinations of the distribution function for particle size and of its parameters were then used for correcting the error in the estimate of the critical size from the activity found in supernatants from centrifugations in fixed angle rotors.     

Comparative studies on membrane proteins of Nitrobacter hamburgensis and Nitrobacter winogradskyi

Abstract The inner membranes (i.e. cytoplasmic and intracytoplasmic membranes, IM) of autotrophically, mixotrophically, and heterotrophically grown cells of Nitrobacter hamburgensis were characterized with respect to their nitrite oxidase activity, density, and protein pattern as revealed by two-dimensional gel electrophoresis. In contrast to the IM of heterotrophically grown cells, those of auto- and mixotrophically grown cells had a high nitrite oxidase activity, a higher density of 1.18 g · cm⁻³, and the M_r values of the three typical major proteins were 32 000, 70 000, and 116 000, respectively. The 32-kDa protein contained haem c. The IM of mixotrophically grown cells of Nitrobacter winogradskyi were similar to those of N. hamburgensis with respect to the three major proteins, but differed in some other proteins.     

Purification and characterization of ATPase from Nitrobacter winogradskyi

An ATPase was purified from Nitrobacter winogradskyi, and some of its molecular and enzymatic properties were determined. The enzyme was composed of two subunits of 64 and 59 kDa, respectively. The enzyme had its pH optimum at 9.5 and showed a specific activity of 7 units per mg protein. This activity was about 14% and 18% of that of F1-ATPases obtained from Escherichia coli and Sulfolobus acidocaldarius, respectively. The enzyme was 29% and 6% inhibited by 100 microM dicyclohexylcarbodiimide (DCCD) and 100 microM NaN3, respectively. It was not inhibited by 20 mM NaNO3.     

A new facultatively nitrite oxidizing bacterium,Nitrobacter vulgaris sp. nov.

A total of 17 facultatively lithoautotrophic strains of Nitrobacter were investigated. They all were found to be related on the species level by DNA hybridizations. The G+C content of DNA ranged between 58.9 and 59.9 mol %. The isolates originated from divers environments. The cells were 0.5–0.8×1.2–2.0 m in size and motile by one polar to subpolar flagellum. Cell-division normally occurred by budding. Polar caps of intracytoplasmic membranes as well as carboxysomes were present. The cells tended to excrete extracellular polymers forming aggregates or biofilms. Heterotrophic growth was slower than mixotrophic but often faster than litoautotrophic growth. In the presence of nitrite and organic substances the organisms often showed diphasic growth. First nitrite and then the organic material was oxidized. In the absence of oxygen growth was possible by dissimilatory nitrate reduction. Nitrite, nitric and nitrous oxide as well as ammonia were formed. Depending on growth conditions the generation times varied from 12 to 140 h. The new Nitrobacter spec. may be one of the most abundant nitrite-oxidizing bacteria in soils, fresh waters and natural as well as artificial stones. For this organism the name Nitrobacter vulgaris is proposed.The type strain is filed with the culture collection of the Institut für Allgemeine Botanik, Universität Hamburg, FRG.     

Molar growth yield of Nitrobacter winogradskyi during exponential growth

A numerical analysis of experimental growth curves obtained for Nitrobacter by observing changes in cell numbers, substrate concentration and rate of heat evolution has allowed the calculation of the growth rate constants during the phase of balanced growth. The molar growth yield was smaller during that phase than during the phase preceding it. On the other hand, the rate of heat evolution was larger during exponential growth by a factor of about 1.5 than during the stages up to and including this phase. The two observations being in agreement since, if less efficient synthesis occurs during exponential growth, more free energy must be dissipated as heat.     

Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris.

Three monoclonal antibodies (MAbs) against nitrite oxidoreductase (NOR) of Nitrobacter hamburgensis were produced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of the purified enzyme showed that the MAbs named Hyb 153.1 and Hyb 153.3 both recognized a protein with a molecular mass of 64,000 Da, while Hyb 153.2 recognized a protein with a molecular mass of 115,000 Da. The molecular masses of these proteins are in the same range as those of the proteins of the alpha (115,000-Da) or beta (65,000-Da) subunit of the NOR. By using the antibodies, the amount of NOR was shown to be dependent on the growth conditions. The highest level of NOR was observed in N. hamburgensis when cells were growing mixotrophically. Analysis of whole-cell extracts of N. hamburgensis, N. winogradskyi, and N. vulgaris indicated serological homology of the NORs from these species of the genus Nitrobacter. The immunological analysis enables detection of the key enzyme of the genus Nitrobacter.     

Morphologische und physiologische Untersuchungen an Zellen von Nitrobacter winogradskyi Buch

Zusammenfassung Wir untersuchten elektronenmikroskopisch das periphere Membransystem und die Polyphosphatgranula in Schnitten von Nitrobacter winogradskyi und verglichen beide mit physiologischen Daten. Die Nitrit-Oxydationsaktivität ist abhängig von der Konzentration des Cytochroms c. Ganz allgemein haben Zellen mit einer hohen Aktivität eine große, solche mit einer niedrigen eine kleine Cytochrommenge. Entsprechend ändert sich der Membrangehalt. Aktive Zellen mit hohem Cytochromgehalt enthalten viele Membranen, inaktive mit geringem Gehalt nur wenige. Außerdem ist die Aktivität pro Cytochrommolekül nicht konstant. Zellen, die lange Zeit ohne Nitrit gestanden haben, besitzen eine niedrige, solche aus der logarithmischen Wachstumsphase eine hohe Umsatzrate. Die Ursache ist nicht bekannt. Die Zahl der Polyphosphatgranula steigt mit zunehmendem Gehalt an Polyphosphaten in der Bakterienzelle.

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Morphological and physiological investigations in Nitrobacter winogradskyi Buch

Summary The peripheral membrane system and polyphosphate granules of Nitrobacter winogradskyi as they are revealed by sections studied in the electron microscope are regarded in relation to physiological data. The activity of nitrite oxydation depends on the concentration of cytochrome c. Cells being highly active generally show a relatively high amount of cytochrome c, whereas this is not the case in less active cells. The number of membrane pairs is affected simultaneously: active cells of high cytochrome concentration contain a high number of membrane pairs; those which are less active and have a lower cytochrome concentration, possess only a few membrane pairs. In addition the activity per mol cytochrome varies. Cells having been short of nitrite for a long time show a low turnover rate, whereas in the logarithmic phase of growth the turnover rate is considerably higher. The reason of this is not yet known. The number of polyphosphate granules increases in relation to the amount of polyphosphate found in the bacterial cell.

     

Genome Sequence of the Chemolithoautotrophic Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Nb-255

The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C₂ to C₄ metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C₆ molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.     

Energy metabolism of autotrophically and heterotrophically grown cells of Nitrobacter winogradskyi

The adenine nucleotide pools and the NADH pool were compared in intact Nitrobacter winogradskyi cells grown under different conditions. The NADH pool was highest in nitrite-grown cells (22.0 nmol/mg N), less high in acetategrown cells (15.1 nmol/mg N),and lowest in pyruvate-grown cells (11.9 nmol/mg N).The adenine nucleotide pools and the NADH pool were determined after the transition from anaerobic to aerobic conditions.In both autotrophically and heterotrophically grown cells the ATP pool decreased within the first second after the addition of oxygen and then increased.In cells grown with nitrite or acetate the NADH pool increased the first second after the addition of oxygen then decreased below the initial value. In pyruvate-grown cells the changes in the NADH pool were less obvious.In the presence of rotenone autotrophic cells were able to generate ATP, but the reverse energy-dependent electron transport was inhibited. Consequently, NADH was not synthesized. N,N-dicyclohexylcarbodiimide an inhibitor of ATPase, prevented both ATP and NADH generation.Abbreviations DCCD N,N-dicyclohexylcarbodiimide