Hans-Peter Koops

Phylogeny of All Recognized Species of Ammonia Oxidizers Based on Comparative 16S rRNA and amoA Sequence Analysis: Implications for Molecular Diversity Surveys

ABSTRACT The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoAlevel. In this study, the 16S rDNA sequence data of 10Nitrosomonas species and Nitrosococcus mobiliswere completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoAsequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.

In situ Identification of Ammonia-oxidizing Bacteria

Summary A 16S rRNA-targeted oligonucleotide probe (NEU) specific for some representatives of the lithoautotrophic ammonia-oxidizing bacteria was developed based on comparative sequence analysis. Whole cell hybridization of target cells in combination with digital microscopy was used to determine the optimal hybridization stringency. In situ hybridization of several activated sludge samples and a trickling filter biofilm with probe NEU allowed the detection of dense cell clusters exclusively in those samples originating from sewage treatment plants with stable nitrification. Scanning confocal laser microscopy revealed that these aggregates were built up by up to 3,000 cells. Specific arrangements of intracytoplasmic membranes detected in these clusters in activated sludge ultrathin sections by transmission electromicroscopy independently confirmed the presence of ammonia-oxidizing bacteria. In activated sludge samples of the animal waste processing treatment plant Kraftisried up to 20% of total bacteria could be identified in situ as ammonia-oxidizers. The specific activity (k o ) per in situ detected ammonia-oxidizing cell was calculated to be in the range of 0.00022 ± 0.000045 pmol/cell/hr. Allylthiourea and sodium chlorate were used for specific inhibition of autotrophic ammonia- and nitrite-oxidiziers in samples of the plant Kraftisried. No hcterorrophic nitrification was detected. Inhibition of ammonia-oxidizing bacteria over a period of five hours did not result in a measurable decrease of the cellular rRNA content.

Phylogenetic Diversity within the Genus Nitrosomonas

Summary The phylogenetic relationships within the genus Nitrosomonas were examined in order to obtain a basis for the development of a taxonomically, as well as ecophysiologically related gene probe system suitable for in situ analysis of natural populations of ammonia-oxidizing bacteria. In addition to the 10 validly described species and three undefined species of the genus Nitrosomonas, Nitrosococcus mobilis was included in this study. On the basis of 16S rRNA gene sequence results and DNA-DNA hybridization data, six distinct lines of descent, containing closely related or single species, can be distinguished. One of these lineages allied N. europaea and N. eutropha , which are both common in eutrophic freshwater habitats and which are both halotolerant, together with the obligately halophilic N. halophila . All these three species are urease negative. A second lineage was defined by the three marine species N. marina, N. aestuarii and Nitrosomonas sp. Nm 51, which are all obligately halophilic and urease positive. Two other distinct groups comprised oligotrophic Nitrosomonas species originating from terrestrial or freshwater environments. Members of one group were N. ureae and N. oligotropha , both being urease positive, and the other group contained the urease positive N. nitrosa together with the urease negative N. communis and Nitrosomonas spp. Nm 33 and Nm 41. The remaining two lineages were represented by single marine species, N. cryotolerans and Nitrosococcus mobilis , respectively. Based on currently available 16S rDNA sequences primer pairs useful for specific PCR assisted detection of ecophysiologically defined groups of ammonia oxidizers were selected.

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 μm 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.

Deoxyribonucleic acid homologies among 96 strains of ammonia-oxidizing bacteria

DNA of 96 strains of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio was isolated and analysed spectrophotometrically. Percentages of guanine plus cytosine (G+C) content, genome sizes, and DNA-DNA homologies were determined. The results indicated the presence of eight Nitrosomonas species, three or four Nitrosococcus species, five Nitrosospira species, and two species of both Nitrosolobus and Nitrosovibrio. DNA homologies between strains of a separate species ranged from 56–100%. Average homologies between strains of different species were 33% in Nitrosococcus, 36% in Nitrosomonas, 37% in Nitrosolobus, 40% in Nitrosospira, and 42% in Nitrosovibrio. Average homologies between species of different genera were 33% and thus not significantly above the background value of 30% detected between DNA of ammonia-oxidizing bacteria and Escherichia coli. Genome sizes ranged from 1.90–2.74×109 dalton in Nitrosomonas, 2.09–2.37×109 dalton in Nitrosococcus, 1.87–2.15×109 dalton in Nitrosospira, 1.92–2.10×109 dalton in Nitrosolobus, and 1.91–2.15×109 dalton in Nitrosovibrio. Differences in genome sizes were in accordance with DNA homologies.

Description of a new species of Nitrosococcus

A new species of Nitrosococcus is described. It resembles Nitrosococcus oceanus in shape, size, and ultrastructure of the cells. However, the new species has a more pronounced salt requirement, corresponding to its natural habitats. Two strains were isolated from a salt lake in Saudi Arabia and a salt lagoon in the Mediterranean Sea, respectively. In contrast to N. oceanus, both isolates of the new species were unable to utilize urea as ammonia source. Both species also differed in gelelectrophoretic cell protein patterns. The name N. halophilus is proposed.

Exopolymers: An ecological characteristic of a floc-attached, ammonia-oxidizing bacterium

A lithotrophic ammonia-oxidizing bacterium of the Nitrosomonas type was isolated from the lower River Elbe. Enrichment was attained from suspended particulate matter (SPM) of a water sample. At its natural environment, this species almost exclusively occurred attached to flocs, as demonstrated with the immunofluorescence technique. On the species level, the isolate was not related to any of the described Nitrosomonas species. The strain was characterized by strong production of exopolymeric substances (EPS) and was observed to occur self-flocculating in pure cultures. Low ammonia concentrations stimulated EPS production. The EPS revealed an extensive capacity for binding particulate and dissolved materials, as well as cells of other bacterial species. This capacity was affected by changing pH values or salt concentrations of the medium. The EPS appeared to function as a buffer against toxic compounds and against changing environmental conditions. Another Nitrosomonas strain isolated from the Elbe estuary, but lacking recognizable EPS production, was used for comparison.

Isolation of a moderate halophilic ammonia-oxidizing bacterium, Nitrosococcus mobilis nov. sp.

An ammonia-oxidizing bacterium was isolated from a sample of brackish water (North Sea, Harbour of Husum). It is a motile large coccus 1.5–1.7 μm in diameter. The extensive cytomembrane system occurring as flattened vesicles in the peripheral region of the cytoplasm and as intrusions into the center of the cytoplasm is to be emphasized as a characteristic mark of identification. The lithoauto-trophically growing bacterium turned out to be an obligate halophile. Because of its physiological and morphological properties, we assigned it to the genus Nitrosoccus and propose the name Nitrosococcus mobilis.

An ammonia-oxidizing bacterium, Nitrosovibrio tenuis nov. gen. nov. sp.

An ammonia-oxidizing, autotroph growing, slender, curved rod was isolated from the soil of Hawaii. It is well distinguishable from any other nitrifying bacteria thus far described by their morphology. The cells are 1.1–3.0 μm long and 0.3–0.4 μm wide. They are motile by means of 1–4 subpolar to lateral flagella. In contrast to most of the ammonia-oxidizing bacteria the isolated vibrio is void of an extensive cytomembrane system. To categorize this not yet described species we propose to create the new genus Nitrosovibrio and to classify the isolated strain as Nitrosovibrio tenuis.

Incorporation of organic compounds into cell protein by lithotrophic, ammonia-oxidizing bacteria

Incorporation of organic compounds into cell protein by the obligate chemolithotrophs Nitrosomonas spec., Nitrosococcus oceanus, Nitrosococcus mobilis, Nitrosovibrio tenuis, Nitrosolobus spec., and Nitrosopira spec. was studied. In the presence of ammonia as energy source organic substrates were supplied. Distribution of 14C into cell amino acids arising from 14C-labelled glucose, Na-pyruvate, and Na-acetate was investigated. While carbon from glucose was distributed unrestricted, carbon from pyruvate preferably entered into the amino acids of the pyruvate and glutamate family and from acetate mainly into leucine and the glutamate family. Among the strains examined, slight differences were observed, but all should be included under group A of the scheme of Smith and Hoare (1977).