Temperature influences the population structure of nitrite-oxidizing bacteria in activated sludge.

Activated sludge from the municipal waste water treatment plant in Hamburg was seeded with mineral nitrite medium and incubated at 10°C, 17°C and 28°C. Dominant lithoautotrophic nitrite-oxidizing bacteria have been identified by electron microscopy, denaturing and temperature gradient gel electrophoresis and PCR with genus-specific primer pairs. The results have revealed the existence of three different genera of nitrite-oxidizing bacteria, namely Nitrospira, Nitrobacter and a novel cold-adapted nitrite oxidizer. As shown by electron microscopy members of the novel genus coexisted in activated sludge together with Nitrospira. A temperature-dependent shift in the population structure was demonstrated by cultivation-based approaches. The novel nitrite oxidizer was enriched at temperatures of 10°C and 17°C. Representatives of Nitrospira were able to grow in a broad temperature range between 10°C and 28°C and members of Nitrobacter were enriched during incubations at 17°C and 28°C. By subsequent 16S rDNA sequencing, the cold-adapted nitrite oxidizer was shown to be closely related to the betaproteobacterium 'Candidatus Nitrotoga arctica'. These findings demonstrated that the population structure of nitrite-oxidizing bacteria in activated sludge is more complex than previously thought and responds strongly to long-term temperature changes.

Physiological and phylogenetic characterization of a novel lithoautotrophic nitrite-oxidizing bacterium, 'Candidatus Nitrospira bockiana'.

A new isolate of a lithoautotrophic nitrite-oxidizing bacterium was obtained from internal corrosion deposits from a steel pipeline of the Moscow heating system. The organism oxidized nitrite as the sole energy source and fixed carbon dioxide as the only carbon source. The cells were extremely pleomorphic: loosely wound spirals, slightly curved and even straight rods were detected, as well as coccoid cells. The highest rate of nitrite consumption (1.5 mM nitrite as substrate) was measured at 42 degrees C, with a temperature range of 28-44 degrees C. In enrichment cultures with Nocardioides sp. as an accompanying organism, optimal oxidation of 5.8 mM nitrite occurred at 45 degrees C, with a range of 28-48 degrees C. Neither pyruvate nor yeast extract stimulated nitrification. Organotrophic growth was not observed. Phylogenetic analysis of 16S rRNA gene sequences revealed that the novel isolate represents a new sublineage of the genus Nitrospira. On the basis of physiological, chemotaxonomic and molecular characteristics, the name 'Candidatus Nitrospira bockiana' is proposed.

Immunological detection of Nitrospira-like bacteria in various soils.

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.

Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity.

The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter, Nitrococcus, Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis-9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis-9-hcxadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis-7 and cis-11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.

Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase.

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated that Nitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS.

Isolation and immunocytochemical location of the nitrite-oxidizing system in nitrospira moscoviensis

A membrane-associated nitrite-oxidizing system of Nitrospira moscoviensis was isolated from heat-treated membranes. The four major proteins of the enzyme fraction had apparent molecular masses of 130, 62, 46, and 29 kDa, respectively. The nitrite-oxidizing activity was dependent on the presence of molybdenum. In contrast to the nitrite oxidoreductase of Nitrobacter hamburgensis X14, the activity of the nitrite-oxidizing system of Ns. moscoviensis increased when solubilized by heat treatment. Electron microscopy of the purified enzyme revealed uniform particles with a size of approximately 7 x 9 nm. SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153-3, which recognize the beta-subunit of the nitrite oxidoreductase from Nitrobacter, reacted with a protein of 50 kDa in Ns. moscoviensis. This protein corresponded to the protein of 46 kDa of the purified enzyme and contained a b-type cytochrome. Using electron microscopic immunocytochemistry and the monoclonal antibodies Hyb 153-3, the nitrite-oxidizing system of Ns. moscoviensis was shown to be located in the periplasmic space. Here a periodic arrangement of membrane-associated particles was found on the outside of the cytoplasmic membrane in the form of a hexagonal pattern. It is supposed that these particles represent the nitrite-oxidizing system in Nitrospira.

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.