Global distribution and diversity of coral-associated Archaea and their possible role in the coral holobiont nitrogen cycle

Diversity, distribution and genetic comparison of Archaea associated with the surface mucus of corals from three genera, namely Acanthastrea sp., Favia sp. and Fungia sp., from the Gulf of Eilat, Israel and from Heron Island, Australia were studied. Sequencing of the 16S rRNA gene of the coral-associated Archaea revealed dominance of Crenarchaeota (79%, on average). In this phylum, 87% of the sequences were similar (>or= 97%) to the Thermoprotei, with 76% of these being similar (>or= 97%) to the ammonium oxidizer, Nitrosopumilus maritimus. Most of the coral-associated euryarchaeotal sequences (69%) were related to marine group II, while other euryarchaeotal clades were found to be related to anaerobic methanotrophs (8%), anaerobic nitrate reducers (i.e. denitrification, 15%) and marine group III (8%). Most of the crenarchaeotal and euryarchaeotal coral-associated 16S rRNA gene sequences from Heron Island (61%) and from the Gulf of Eilat (71%) were closely related (>or= 97%) to sequences previously derived from corals from the Virgin Islands. Analysis of archaeal amoA sequences obtained from the fungiid coral, Fungia granulosa, divided into three clades, all related to archaeal sequences previously obtained from the marine environment. These sequences were distantly related to amoA sequences previously found in association with other coral species. Preliminary experiments suggest that there is active oxidation of ammonia to nitrite in the mucus of F. granulosa. Thus, coral-associated Archaea may contribute to nitrogen recycling in the holobiont, presumably by acting as a nutritional sink for excess ammonium trapped in the mucus layer, through nitrification and denitrification processes.     

Molecular phylogeny of the Robust clade (Faviidae, Mussidae, Merulinidae, and Pectiniidae): An Indian Ocean perspective

Recent phylogenetic analyses have demonstrated the limits of traditional coral taxonomy based solely on skeletal morphology. In this phylogenetic context, Faviidae and Mussidae are ecologically dominant families comprising one third of scleractinian reef coral genera, but their phylogenies remain partially unresolved. Many of their taxa are scattered throughout most of the clades of the Robust group, and major systematic incongruences exist. Numerous genera and species remain unstudied, and the entire biogeographic area of the Indian Ocean remains largely unsampled. In this study, we analyzed a portion of the mitochondrial cytochrome c oxidase subunit 1 gene and a portion of ribosomal DNA for 14 genera and 27 species of the Faviidae and Mussidae collected from the Indian Ocean and New Caledonia and this is the first analysis of five of these species. For some taxa, newly discovered evolutionary relationships were detected, such as the evolutionary distinctiveness of Acanthastrea maxima, the genetic overlap of Parasimplastrea omanensis and Blastomussa merleti, and the peculiar position of Favites peresi in clade XVII together with Echinopora and Montastraea salebrosa. Moreover, numerous cases of intraspecific divergences between Indian Ocean and Pacific Ocean populations were detected. The most striking cases involve the genera Favites and Favia, and in particular Favites complanata, F. halicora, Favia favus, F. pallida, F. matthaii, and F. rotumana, but divergence also is evident in Blastomussa merleti, Cyphastrea serailia, and Echinopora gemmacea. High morphological variability characterizes most of these taxa, thus traditional skeletal characteristics, such as corallite arrangement, seem to be evolutionary misleading and are plagued by convergence. Our results indicate that the systematics of the Faviidae and the Mussidae is far from being resolved and that the inclusion of conspecific populations of different geographical origin represents an unavoidable step when redescribing the taxonomy and systematics of scleractinian corals. More molecular phylogenies are needed to define the evolutionary lineages that could be corroborated by known and newly discovered micromorphological characters.     

丛生盔形珊瑚共附生可培养真菌多样性分析

旨在研究珊瑚共附生可培养真菌多样性。运用稀释平板法和基于ITS-rDNA基因序列的系统发育分析对珊瑚共附生可培养真菌多样性进行研究, 将所得到的基因序列与NCBI数据库GenBank中的序列进行相似性比较并构建系统发育树。实验中从丛生盔形珊瑚表面和内部共分离得到19株菌落形态各异的真菌。ITS-rDNA序列分析及形态学鉴定表明, 丛生盔形珊瑚共附生真菌主要包括曲霉菌Aspergillus sp.、枝孢霉菌Cladosporium sp.、炭角菌Xylariales sp.、青霉菌Penicillium sp.、葡萄穗霉菌Stachybotrys sp.、赤霉菌Gibberella moniliformis、镰刀霉菌Fusarium sp.等。分离得到的菌株中, 4-13与GenBank中已报道的基因序列的相似性仅为89%。结果表明, 与丛生盔形珊瑚共附生的可培养真菌较为丰富, 是潜在的新的微生物菌种资源, 具有进一步研究的价值。     

Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes

The annual changes in the composition and abundance of ammonia-oxidizing archaea (AOA) were analyzed monthly in surface waters of three high mountain lakes within the Limnological Observatory of the Pyrenees (LOOP; northeast Spain) using both 16S rRNA and functional (ammonia monooxygenase gene, amoA) gene sequencing as well as quantitative PCR amplification. The set of biological data was related to changes in nitrogen species and to other relevant environmental variables. The whole archaeal assemblage was dominated by phylotypes closely related to the crenarchaeal 1.1a group (58% ± 18% of total 16S rRNA gene sequences), and consistent structural changes were detected during the study. Water temperature was the environmental variable that better explained spring, summer, and winter (ice-covered lakes) archaeal assemblage structure. The amoA gene was detected year round, and seasonal changes in amoA gene composition were well correlated with changes in the archaeal 16S rRNA gene pool. In addition, copy numbers of both the specific 1.1a group 16 rRNA and archaeal amoA genes were well correlated, suggesting that most freshwater 1.1a Crenarchaeota had the potential to carry out ammonia oxidation. Seasonal changes in the diversity and abundance of AOA (i.e., amoA) were better explained by temporal changes in ammonium, the substrate for nitrification, and mostly nitrite, the product of ammonia oxidation. Lacustrine amoA gene sequences grouped in coherent freshwater phylogenetic clusters, suggesting that freshwater habitats harbor typical amoA-containing ecotypes, which is different from soils and seas. We observed within the freshwater amoA gene sequence pool a high genetic divergence (translating to up to 32% amino acid divergence) between the spring and the remaining AOA assemblages. This suggests that different AOA ecotypes are adapted to different temporal ecological niches in these lakes.     

Favia camranensis sp. n. (Scleractinia: Faviidae), a new coral species from Southern Vietnam

A new species of coral, Favia camranensis sp. n., from the Hon Nai reef, Cam Ranh Bay, in southern Vietnam is described. This coral differs from all known faviids in that its corallites project one above another by more than 2 mm. The septa and septa-costae are heavily ornamented with complexly serrated trabecular spines. This coral can form monospecific aggregations of 5–20 colonies.     

Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

Comparative analysis of archaeal 16S rRNA and <Emphasis Type="Italic">amoA</Emphasis> genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1 x 10(7) to 4.9 x 10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.     

Communities of archaea and bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps, and evidence of ammonia-oxidizing Crenarchaeota

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.     

Bacteria associated with an encrusting sponge (Terpios hoshinota) and the corals partially covered by the sponge

Terpios hoshinota, a dark encrusting sponge, is known to be a competitor for space in coral reef environments, and facilitates the death of corals. Although numerous cyanobacteria have been detected in the sponge, little is known of the sponge-associated bacterial community. This study examined the sponge-associated bacterial community and the difference between the bacterial communities in the sponge and the coral partially covered by the sponge by analysis of 16S rRNA gene sequences of samples isolated from the sponge covering the corals Favia complanata, Isopora palifera, Millepora sp., Montipora efflorescens and Porites lutea. The sponge-associated bacterial community was mainly (61-98%) composed of cyanobacteria, with approximately 15% of these alphaproteobacteria and gammaproteobacteria, although the proportions varied in different sponge samples. The dominant cyanobacteria group was an isolated group closely related to Prochloron sp. The comparison of the bacterial communities isolated from sponge-free and the sponge-covered P. lutea showed that covering by the sponge caused changes in the coral-associated bacterial communities, with the presence of bacteria similar to those detected in black-band disease, suggesting the sponge might benefit from the presence of bacteria associated with unhealthy coral, particularly in the parts of the coral closest to the margin of the sponge.     

Vertical structure of archaeal communities and the distribution of ammonia monooxygenase A gene variants in two meromictic High Arctic lakes

The distribution of archaeal amoA and 16S rRNA genes was evaluated in two marine-derived, meromictic lakes in the Canadian High Arctic: Lake A and Lake C1 on the northern coast of Ellesmere Island. The amoA gene was recorded in both lakes, with highest copy numbers in the oxycline. Sequence analysis showed that amoA from the two lakes shared 94% similarity, indicating at least two phylogenetically distinct clusters. Clone libraries of archaeal 16S rRNA genes from Lake A revealed strong vertical differences in archaeal community diversity and composition down the water column. The oxic layer was dominated by one group of Euryarchaeota affiliated to the Lake Dagow Sediment (LDS) cluster. This group was absent from the oxycline, which had an extremely low archaeal diversity of two phylotypes. Both belonged to the Crenarchaeota Marine Group I (MGI), the marine group that has been linked to archaeal amoA ; however, there was a low ratio of amoA to MGI copy numbers, suggesting that many MGI Archaea did not carry the amoA gene. The anoxic zone contained representatives of the RC-V (Rice Cluster-V) and LDS clusters of Euryarchaeota. These results show the strong vertical differentiation of archaeal communities in polar meromictic lakes, and they suggest archaeal nitrification within the oxycline of these highly stratified waters.     

Early Silurian(Telychian)rugose coral fauna of Daguan area, northeast Yunnan Province,China

Study on rugose coral fauna of the Sifengya Formation(early Telyehian)and Daluzhai Formation (mid-late Telychian)in Daguan area,northeast Yunnan Province,China was carded out.Rugose coral fauna of the Sifengya Formation included 18 genera and 34 species,while Daluzhai Formation with nine genera,ten species.We described rugose coral fauna(12 genera,19 species)including one new genus and five new species,i.e.Protoketophyllum daguanense gen.et sp.nov..Crassilasma huanggexiense sp.nov.,Pseudophaulactis heae sp.nov.,P.convolutus sp.nov.,and Shensiphyllum minor sp.nov..The characteristics and geological significance of rugose coral fauna of Sifengya Formation and Daluzhai Formation were analyzed.Particularly,mgose coral fauna of the Sifengya Formation represent early Telychian rugosan fauna in the Upper Yangtze region and improve the sequences of early Silurian(Llandovery)mgose coral assemblages in Yangtze region.It is therefore very meaningful to further analyze radiation period of rugose coral fauna in such epoch.     

Ammonia-oxidizing bacteria along meadow-to-forest transects in the Oregon Cascade Mountains

Although nitrification has been well studied in coniferous forests of Western North America, communities of NH(3)-oxidizing bacteria in these forests have not been characterized. Studies were conducted along meadow-to-forest transects at two sites (Lookout and Carpenter) in the H. J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon. Soil samples taken at 10- or 20-m intervals along the transects showed that several soil properties, including net nitrogen mineralization and nitrification potential rates changed significantly between vegetation zones. Nonetheless, terminal restriction fragment length polymorphism (T-RFLP) analysis of the PCR-amplified NH(3) monooxygenase subunit A gene (amoA) showed the same DNA fragments (TaqI [283 bp], CfoI [66 bp], and AluI [392 bp]) to dominate >/=45 of 47 soil samples recovered from both sites. Two fragments (491-bp AluI [AluI491] and CfoI135) were found more frequently in meadow and transition zone soil samples than in forest samples at both sites. At the Lookout site the combination AluI491-CfoI135 was found primarily in meadow samples expressing the highest N mineralization rates. Four unique amoA sequences were identified among 15 isolates recovered into pure culture from various transect locations. Six isolates possessed the most common T-RFLP amoA fingerprint of the soil samples (TaqI283-AluI392-CfoI66), and their amoA sequences shared 99.8% similarity with a cultured species, Nitrosospira sp. strain Ka4 (cluster 4). The other three amoA sequences were most similar to sequences of Nitrosospira sp. strain Nsp1 and Nitrosospira briensis (cluster 3). 16S ribosomal DNA sequence analysis confirmed the affiliation of these isolates with Nitrosospira clusters 3 and 4. Two amoA clone sequences matched T-RFLP fingerprints found in soil, but they were not found among the isolates.     

Identification of coral endosymbionts of Veedhalai and Mandapam coasts of Palk Bay,India using small subunit rDNA

Coral endosymbionts act as a bio-indicator of coral ecosystem under extreme environmental conditions. The health of the coral ecosystem depends on the endosymbiont cell density of the coral hosts. Therefore, it is of interest to analyze ten coral fragments found to be under the genera Acropora, Favites, Favia, and Porites collected at various locations from Veedhalai to Mandapam, southeast coast of India during January 2019 to March 2019. The zooxanthellae cell count ranged between 4.08 (Porites sp.9) and 13.75x105 cells cm2 -1 (Favites sp.3). This indicates the health of the corals in the region. The genus (clade) level identification of endosymbionts was detected using the host excluding primers of small subunit DNA (nssrDNA). Bidirectional sequencing of 18S nrDNA gene (SSU) of all ten coral fragments show that the Veedhalai corals is associated with the genus Durusdinium (Clade D) but the corals of Mandapam is associated with the genera, Cladocopium (Clade C) and Durusdinium (Clade D). It is known that the thermal stress has negative impact on coral reef ecosystem of the world. The dominance of the genus Durusdinium in the scleractinian corals of Palk Bay may be due to frequent exposure to thermal stress. This thermotolerant endosymbionts is opportunistic. Thus, the corals of Veedhalai and Mandapam coasts, Palk Bay, India are necessarily packed with thermotolerant endosymbionts enabling conservation.     

amoA-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions

Ammonia-oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species-level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species-level and represented 30-37 additional species-level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8-83 co-existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.     

Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.     

Re-evaluation of the systematics of the endemic corals of Brazil by molecular data

Recent genetic work on various coral genera has shown that morphological convergence between Atlantic and Pacific corals obscures evolutionary relationships and inferred levels of endemicity between the regions. Based on DNA sequences from nuclear and mitochondrial loci that provide higher resolution than those previously presented, this study shows that relationships within parts of the Atlantic coral fauna are also in need of substantial revision. The data presented here indicate that (1) the endemic Brazilian genus Mussismilia is a monophyletic clade, (2) Mussismilia is more closely related to the Caribbean Faviidae than Mussidae, the family in which it is currently placed, (3) the Brazilian endemic coral Favia leptophylla is much more closely related to Mussismilia than other species of Favia and has most likely been incorrectly placed in the genus Favia and (4) the other endemic Favia species found in Brazil, Favia gravida, is genetically distinct from Favia fragum, a Caribbean congener with which it is frequently synonymized. The nuclear data also suggest the possible presence of a cryptic species within Mussismilia, but additional sampling and morphological information is required to confirm this finding.     

Early Silurian (Telychian) rugose coral fauna of Daguan area, northeast Yunnan province, China

Study on rugose coral fauna of the Sifengya Formation (early Telychian) and Daluzhai Formation (mid-late Telychian) in Daguan area, northeast Yunnan Province, China was carried out. Rugose coral fauna of the Sifengya Formation included 18 genera and 34 species, while Daluzhai Formation with nine genera, ten species. We described rugose coral fauna (12 genera, 19 species) including one new genus and five new species, i.e. Protoketophyllum daguanense gen. et sp. nov., Crassilasma huanggexiense sp. nov., Pseudophaulactis heae sp. nov., P. convolutus sp. nov., and Shensiphyllum minor sp. nov. The characteristics and geological significance of rugose coral fauna of Sifengya Formation and Daluzhai Formation were analyzed. Particularly, rugose coral fauna of the Sifengya Formation represent early Telychian rugosan fauna in the Upper Yangtze region and improve the sequences of early Silurian (Llandovery) rugose coral assemblages in Yangtze region. It is therefore very meaningful to further analyze radiation period of rugose coral fauna in such epoch. __________ Translated from Acta Palaeontologica Sinica, 2005, 44(2): 229–246 [译自: 古生物学报, 2005, 44(2): 229–246]     

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys

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.