Re-evaluation of the phylogenetic diversity and global distribution of the genus Candidatus Accumulibacter

Candidatus Accumulibacter was the first microorganism identified as a polyphosphate-accumulating organism (PAO), important for phosphorus removal from wastewater. This genus is diverse, and the current phylogeny and taxonomic framework appears complicated, with the majority of publicly available genomes classified as Candidatus Accumulibacter phosphatis, despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer scale differentiation into different types and clades, nevertheless taxonomic assignments remain confusing and inconsistent across studies. Therefore, a comprehensive re-evaluation is needed to establish a common understanding of this genus, both in terms of naming and basic conserved physiological traits. Here, we provide this re-assessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive datasets. We identified 15 novel species, along with the well-known Ca. A. phosphatis, Ca. A. deltensis and Ca. A. aalborgensis. To compare the species in situ, we designed new species-specific FISH probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, Ca. Accumulibacter species were widespread in WWTPs with phosphorus removal, indicating the process design as a major driver for their abundance. Genome mining for PAO related pathways and FISH-Raman microspectroscopy confirmed the potential for the PAO metabolism in all Ca. Accumulibacter species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed fine-scale differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal.

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Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum

Applied and Environmental Microbiology ◽

10.1128/aem.00067-09 ◽

2009 ◽

Vol 75 (11) ◽

pp. 3656-3662 ◽

Cited By ~ 318

Author(s):

Katharina F. Ettwig ◽

Theo van Alen ◽

Katinka T. van de Pas-Schoonen ◽

Mike S. M. Jetten ◽

Marc Strous

Keyword(s):

Methane Oxidation ◽

Enrichment Culture ◽

Nitrite Reduction ◽

Anaerobic Methane Oxidation ◽

Rrna Gene ◽

Total Biomass ◽

Environmental Distribution ◽

Diverse Community ◽

Reducing Activity

ABSTRACT Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitrite-reducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present.

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Identification of a novel subgroup of uncultured gammaproteobacterial glycogen-accumulating organisms in enhanced biological phosphorus removal sludge

Microbiology ◽

10.1099/mic.0.047779-0 ◽

2011 ◽

Vol 157 (8) ◽

pp. 2287-2296 ◽

Cited By ~ 8

Author(s):

Jeong Myeong Kim ◽

Hyo Jung Lee ◽

Dae Sung Lee ◽

Kangseok Lee ◽

Che Ok Jeon

Keyword(s):

Phosphorus Removal ◽

Batch Reactor ◽

Full Scale ◽

Rrna Gene ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Phenotypic Properties ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

The presence of glycogen-accumulating organisms (GAO) has been hypothesized to be a cause of deterioration in enhanced biological phosphorus removal (EBPR) processes due to their abilities to out-compete polyphosphate-accumulating organisms (PAO). Based on 16S rRNA gene sequences, new members of uncultured gammaproteobacterial GAO (GB) were identified from sludge samples of a lab-scale sequencing batch reactor used for EBPR. The new GB formed a phylogenetic lineage (GB8) clearly distinct from the previously reported seven GB subgroups. Because the new GB8 members were not targeted by the known fluorescence in situ hybridization (FISH) oligonucleotide probes, a GB8-specific FISH probe (GB429) and a new FISH probe (GB742) targeting all eight GB subgroups were designed, and the phenotypic properties of the new GB8 members were investigated. FISH and microautoradiography approaches showed that GB429-targeted cells (GB8) were large coccobacilli (2–4 µm in size) with the ability to take up acetate under anaerobic conditions, but unable to accumulate polyphosphate under the subsequent aerobic conditions, consistent with in situ phenotypes of GB. FISH analyses on several sludge samples showed that members of GB8 were commonly detected as the majority of GB in lab- and full-scale EBPR processes. In conclusion, this study showed that members of GB8 could be a subgroup of GB with an important role in EBPR deterioration. Designs of FISH probes which hybridize with broader GB subgroups at different hierarchical levels will contribute to studies of the distributions and ecophysiologies of GB in lab- or full-scale EBPR plants.

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Variability in Host Specificity and Functional Potential of Antarctic Sponge-Associated Bacterial Communities

Frontiers in Microbiology ◽

10.3389/fmicb.2021.771589 ◽

2022 ◽

Vol 12 ◽

Author(s):

Antonia Cristi ◽

Génesis Parada-Pozo ◽

Felipe Morales-Vicencio ◽

César A. Cárdenas ◽

Nicole Trefault

Keyword(s):

Bacterial Communities ◽

Bacterial Community Composition ◽

Marker Gene ◽

16S Rrna Gene Sequencing ◽

Sponge Species ◽

Rrna Gene ◽

Functional Potential ◽

Species Specific ◽

Functional Profiles ◽

Antarctic Sponges

Sponge-associated microorganisms are essential for sponge survival. They play an important role in recycling nutrients and, therefore, in the maintenance of the ecosystem. These microorganisms are diverse, species-specific, and different from those in the surrounding seawater. Bacterial sponge symbionts have been extensively studied in the tropics; however, little is known about these microorganisms in sponges from high-latitude environments. Sponges can cover up to 80% of the benthos in Antarctica and are crucial architects for the marine food web. In this study, we present analyses of the bacterial symbionts of three sponges: Haliclona (Rhizoniera) sp., Hymeniacidon torquata, and Isodictya kerguelenensis from the Western Antarctic Peninsula (WAP) with the aim to determine variations on the specificity of the bacteria–sponge interactions and potential signatures on their predicted functional profiles. We use high-throughput 16S rRNA gene sequencing of 30 sponge individuals inhabiting South Bay (Palmer Archipelago, WAP) to describe their microbiome taxonomy and diversity and predict potential functional profiles based on this marker gene. Our work shows similar bacterial community composition profiles among the same sponge species, although the symbiotic relationship is not equally conserved among the three Antarctic sponges. The number of species-specific core operational taxonomic units (OTUs) of these Antarctic sponges was low, with important differences between the total abundance accounted for these OTUs. Only eight OTUs were shared between the three sponge species. Analyses of the functional potential revealed that despite the high host–symbiont specificity, the inferred functions are conserved among these microbiomes, although with differences in the abundance of specific functions. H. torquata showed the highest level of intra-specificity and a higher potential of pathways related to energy metabolism, metabolisms of terpenoids and polyketides, and biosynthesis of other secondary metabolites. Overall, this work shows variations in the specificity of the sponge-associated bacterial communities, differences in how hosts and symbionts establish their relations, and in their potential functional capabilities.

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Genomes from Bacteria Associated with the Canine Oral Cavity: a Test Case for Automated Genome-Based Taxonomic Assignment

10.1101/577163 ◽

2019 ◽

Author(s):

David A. Coil ◽

Guillaume Jospin ◽

Aaron E. Darling ◽

Corrin Wallis ◽

Ian J. Davis ◽

...

Keyword(s):

16S Rrna ◽

Oral Cavity ◽

16S Rrna Gene ◽

Marker Gene ◽

Pcr Amplification ◽

Rrna Gene ◽

Test Case ◽

Bacterial Genomes ◽

Taxonomic Assignment ◽

Taxonomic Assignments

AbstractTaxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach was less subjective, and required far less labor. The primary differences in the remaining taxonomic assignments related to proposed taxonomy changes by the GTDB team.

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Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

Microbiology ◽

10.1099/mic.0.28445-0 ◽

2006 ◽

Vol 152 (2) ◽

pp. 419-429 ◽

Cited By ~ 121

Author(s):

Rikke Louise Meyer ◽

Aaron Marc Saunders ◽

Linda Louise Blackall

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

16S Rrna Gene ◽

Phosphorus Removal ◽

Stable Isotope Probing ◽

Metabolic Phenotype ◽

Rrna Gene ◽

Biological Phosphorus Removal ◽

16S Rrna Gene Analysis

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90 % identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic–aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6·6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33 % of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55 % of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

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A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy)

Microorganisms ◽

10.3390/microorganisms8060936 ◽

2020 ◽

Vol 8 (6) ◽

pp. 936 ◽

Cited By ~ 2

Author(s):

Claudia Leoni ◽

Mariateresa Volpicella ◽

Bruno Fosso ◽

Caterina Manzari ◽

Elisabetta Piancone ◽

...

Keyword(s):

Ecological Model ◽

Salinity Range ◽

Rrna Gene ◽

Hypervariable Regions ◽

Cell Counts ◽

Precious Resource ◽

Catalyzed Reporter Deposition ◽

Card Fish ◽

The 16S Rrna Gene

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial “Candidatus Aquiluna” (~19% at 14.5% salinity). Interestingly, “Candidatus Aquiluna” had not been identified before in thalassohaline waters.

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Metabolic pathways inferred from a bacterial marker gene illuminate ecological changes across South Pacific frontal boundaries

Nature Communications ◽

10.1038/s41467-021-22409-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Eric J. Raes ◽

Kristen Karsh ◽

Swan L. S. Sow ◽

Martin Ostrowski ◽

Mark V. Brown ◽

...

Keyword(s):

16S Rrna ◽

Metabolic Pathways ◽

Low Cost ◽

Marker Gene ◽

South Pacific ◽

Rrna Gene ◽

South Pacific Ocean ◽

Bacterial Marker ◽

Gene 16S Rrna ◽

Gene Data

AbstractGlobal oceanographic monitoring initiatives originally measured abiotic essential ocean variables but are currently incorporating biological and metagenomic sampling programs. There is, however, a large knowledge gap on how to infer bacterial functions, the information sought by biogeochemists, ecologists, and modelers, from the bacterial taxonomic information (produced by bacterial marker gene surveys). Here, we provide a correlative understanding of how a bacterial marker gene (16S rRNA) can be used to infer latitudinal trends for metabolic pathways in global monitoring campaigns. From a transect spanning 7000 km in the South Pacific Ocean we infer ten metabolic pathways from 16S rRNA gene sequences and 11 corresponding metagenome samples, which relate to metabolic processes of primary productivity, temperature-regulated thermodynamic effects, coping strategies for nutrient limitation, energy metabolism, and organic matter degradation. This study demonstrates that low-cost, high-throughput bacterial marker gene data, can be used to infer shifts in the metabolic strategies at the community scale.

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Microbial niche differentiation explains nitrite oxidation in marine oxygen minimum zones

The ISME Journal ◽

10.1038/s41396-020-00852-3 ◽

2021 ◽

Author(s):

Xin Sun ◽

Claudia Frey ◽

Emilio Garcia-Robledo ◽

Amal Jayakumar ◽

Bess B. Ward

Keyword(s):

Nitrogen Loss ◽

Niche Differentiation ◽

Nitrite Reduction ◽

Nitrite Oxidation ◽

Fixed Nitrogen ◽

Anoxic Waters ◽

Biogeochemical Models ◽

Nitrite Oxidizing Bacteria ◽

Oxygen Addition ◽

Oxygen Minimum

AbstractNitrite is a pivotal component of the marine nitrogen cycle. The fate of nitrite determines the loss or retention of fixed nitrogen, an essential nutrient for all organisms. Loss occurs via anaerobic nitrite reduction to gases during denitrification and anammox, while retention occurs via nitrite oxidation to nitrate. Nitrite oxidation is usually represented in biogeochemical models by one kinetic parameter and one oxygen threshold, below which nitrite oxidation is set to zero. Here we find that the responses of nitrite oxidation to nitrite and oxygen concentrations vary along a redox gradient in a Pacific Ocean oxygen minimum zone, indicating niche differentiation of nitrite-oxidizing assemblages. Notably, we observe the full inhibition of nitrite oxidation by oxygen addition and nitrite oxidation coupled with nitrogen loss in the absence of oxygen consumption in samples collected from anoxic waters. Nitrite-oxidizing bacteria, including novel clades with high relative abundance in anoxic depths, were also detected in the same samples. Mechanisms corresponding to niche differentiation of nitrite-oxidizing bacteria across the redox gradient are considered. Implementing these mechanisms in biogeochemical models has a significant effect on the estimated fixed nitrogen budget.

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Species-diagnostic and species-specific DNA sequences evenly distributed throughout pine and spruce chromosomes

Genome ◽

10.1139/g10-065 ◽

2010 ◽

Vol 53 (10) ◽

pp. 769-777 ◽

Cited By ~ 1

Author(s):

Melanie Mehes-Smith ◽

Paul Michael ◽

Kabwe Nkongolo

Keyword(s):

Dna Sequences ◽

Random Amplified Polymorphic Dna ◽

Inter Simple Sequence Repeat ◽

Issr Markers ◽

Pinus Monticola ◽

The Family ◽

Species Specific ◽

Rapd And Issr ◽

Specific Sequences

Genome organization in the family Pinaceae is complex and largely unknown. The main purpose of the present study was to develop and physically map species-diagnostic and species-specific molecular markers in pine and spruce. Five RAPD (random amplified polymorphic DNA) and one ISSR (inter-simple sequence repeat) species-diagnostic or species-specific markers for Picea mariana , Picea rubens , Pinus strobus , or Pinus monticola were identified, cloned, and sequenced. In situ hybridization of these sequences to spruce and pine chromosomes showed the sequences to be present in high copy number and evenly distributed throughout the genome. The analysis of centromeric and telomeric regions revealed the absence of significant clustering of species-diagnostic and species-specific sequences in all the chromosomes of the four species studied. Both RAPD and ISSR markers showed similar patterns.

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“Candidatus Dechloromonas phosphoritropha” and “Ca. D. phosphorivorans”, novel polyphosphate accumulating organisms abundant in wastewater treatment systems

The ISME Journal ◽

10.1038/s41396-021-01029-2 ◽

2021 ◽

Author(s):

Francesca Petriglieri ◽

Caitlin Singleton ◽

Miriam Peces ◽

Jette F. Petersen ◽

Marta Nierychlo ◽

...

Keyword(s):

Wastewater Treatment ◽

Wastewater Treatment Plants ◽

Niche Partitioning ◽

16S Rrna Gene Sequencing ◽

Abundant Species ◽

Phosphate Removal ◽

Rrna Gene ◽

Biological Phosphorus Removal ◽

Polyphosphate Accumulating Organisms

AbstractMembers of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.

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