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 amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were 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 amoA sequences 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.Chemolithoautotrophic ammonia-oxidizing bacteria (AOB) play a central role in the natural cycling of nitrogen by aerobically transforming ammonia to nitrite. From an anthropocentric point of view, the activity of AOB is considered to be both detrimental and beneficial. AOB oxidize urea and ammonia fertilizers to nitrite and, in conjunction with nitrite oxidizers which subsequently convert nitrite to nitrate, thus contribute to fertilizer loss from agricultural soils by producing compounds which are easily washed out or used as electron acceptors for denitrification (42). The former process is also responsible for significant pollution of water supplies with nitrite and nitrate. Furthermore, AOB can produce greenhouse gases (8, 74) and corrode, because of the produced acid, stonework and concrete (46). On the other hand, AOB activity is encouraged in wastewater treatment plants to reduce the ammonia content of sewage before discharge into the receiving waters (49). Reduction of ammonia releases into aquatic en...

Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities

“…Bulk genomic DNA was extracted from the aliquots using a series of enzymatic digestions, followed by phenol-chloroform extraction, essentially as described previously 5 . Extracts were then suspended in TE (10 mM Tris, 1 mM EDTA, pH 7.6) and digested with RNase A (10 mg L -1 final concentration) at 37 o C for 30 min.…”

“…These polyphosphate-accumulating organisms (PAOs) are then allowed to settle in a separate tank (clarifier), leaving the effluent water largely Pi-depleted. EBPR is more economical in the long term 2 and has a lower environmental impact 4 than traditional (chemical) Pi removal 5 , but is prone to unpredictable failures due to loss or reduced activity of microbial populations responsible for Pi removal 6 . This is primarily because the design process is highly empirical due to an incomplete understanding of sludge microbial ecology.…”

“…This is primarily because the design process is highly empirical due to an incomplete understanding of sludge microbial ecology. Environmental engineers and microbiologists have been studying EBPR since its introduction in municipal wastewater treatment plants over thirty years ago 5 with the goal of making it a more reliable industrial process. Typically, EBPR is studied in lab-scale sequencing batch reactors (SBRs) where the microbial community can be better monitored and perturbed, and PAOs can be enriched to much higher levels than in full scale systems 7 .…”

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

“…Afterward, they were incubated with the 16S rRNA directed oligonucleotide probe EUB338, Nso 190, NEU, or Nsv 443 in hybridization buffer (0.9 M NaCl, 20 mM M Tris/HCl pH 7.2, 0.01% SDS) with a final concentration of 30% formamide (EUB338, Nsv 443) or 40% formamide (NEU, Nso 190) at a temperature of 46°C for 2 h. As a competitor for NEU, an equimolar amount of unlabeled oligonucleotide CTE was added. Probe EUB338 was used to detect members of the domain Bacteria [12], and probe Nso 190 detects all ammonia oxidizers of the b-subclass of Proteobacteria [35] except some Nitrosomonas strains (Table 1, [40]). Probe NEU is specific for the detection of only a few members of the genus Nitrosomonas (Table 1 [40,50]).…”

“…Probe EUB338 was used to detect members of the domain Bacteria [12], and probe Nso 190 detects all ammonia oxidizers of the b-subclass of Proteobacteria [35] except some Nitrosomonas strains (Table 1, [40]). Probe NEU is specific for the detection of only a few members of the genus Nitrosomonas (Table 1 [40,50]). Probe Nsv 443 is directed to Nitrosolobus multiformis, Nitrosospira briensis and Nitrosovibrio tenuis (Table 1 [35,40]).…”