Taxonomic Re-Classification and Expansion of the Phylum Chloroflexota Based on over 5000 Genomes and Metagenome-Assembled Genomes.

The phylum Chloroflexota (formerly Chloroflexi) encompasses metabolically diverse bacteria that often have high prevalence in terrestrial and aquatic habitats, some even with biotechnological application. However, there is substantial disagreement in public databases which lineage should be considered a member of the phylum and at what taxonomic level. Here, we addressed these issues through extensive phylogenomic analyses. The analyses were based on a collection of >5000 Chloroflexota genomes and metagenome-assembled genomes (MAGs) from public databases, novel environmental sites, as well as newly generated MAGs from publicly available sequence reads via an improved binning approach incorporating covariance information. Based on calculated relative evolutionary divergence, we propose that Candidatus Dormibacterota should be listed as a class (i.e., Ca. Dormibacteria) within Chloroflexota together with the classes Anaerolineae, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Ca. Limnocylindria, Thermomicrobia, and two other classes containing only uncultured members. All other Chloroflexota lineages previously listed at the class rank appear to be rather orders or families in the Anaerolineae and Dehalococcoidia, which contain the vast majority of genomes and exhibited the strongest phylogenetic radiation within the phylum. Furthermore, the study suggests that a common ecophysiological capability of members of the phylum is to successfully cope with low energy fluxes.

Stratiformator vulcanicus gen. nov., sp. nov., a marine member of the family Planctomycetaceae isolated from a red biofilm in the Tyrrhenian Sea close to the volcanic island Panarea.

A novel planctomycetal strain, designated Pan189T, was isolated from biofilm material sampled close to Panarea Island in the Tyrrhenian Sea. Cells of strain Pan189T are round grain rice-shaped, form pink colonies and display typical planctomycetal characteristics including asymmetric cell division through polar budding and presence of crateriform structures. Cells bear a stalk opposite to the division pole and fimbriae cover the cell surface. Strain Pan189T has a mesophilic (optimum at 24 °C) and neutrophilic (optimum at pH 7.5) growth profile, is aerobic and heterotrophic. Under laboratory-scale cultivation conditions, it reached a generation time of 102 h (µmax = 0.0068 h-1), which places the strain among the slowest growing members of the phylum Planctomycetota characterized so far. The genome size of the strain is with 5.23 Mb at the lower limit among the family Planctomycetaceae (5.1-8.9 Mb). Phylogenetically, the strain represents a novel genus and species in the family Planctomycetaceae, order Planctomycetales, class Planctomycetia. We propose the name Stratiformator vulcanicus gen. nov., sp. nov. for the novel taxon, that is represented by the type strain Pan189T (= DSM 101711 T = CECT 30699 T).

How clear is our current view on microbial dark matter? (Re-)assessing public MAG & SAG datasets with MDMcleaner.

As of today, the majority of environmental microorganisms remain uncultured and is therefore referred to as 'microbial dark matter' (MDM). Hence, genomic insights into these organisms are limited to cultivation-independent approaches such as single-cell- and metagenomics. However, without access to cultured representatives for verifying correct taxon-assignments, MDM genomes may cause potentially misleading conclusions based on misclassified or contaminant contigs, thereby obfuscating our view on the uncultured microbial majority. Moreover, gradual database contaminations by past genome submissions can cause error propagations which affect present as well as future comparative genome analyses. Consequently, strict contamination detection and filtering need to be applied, especially in the case of uncultured MDM genomes. Current genome reporting standards, however, emphasize completeness over purity and the de facto gold standard genome assessment tool, checkM, discriminates against uncultured taxa and fragmented genomes. To tackle these issues, we present a novel contig classification, screening, and filtering workflow and corresponding open-source python implementation called MDMcleaner, which was tested and compared to other tools on mock and real datasets. MDMcleaner revealed substantial contaminations overlooked by current screening approaches and sensitively detects misattributed contigs in both novel genomes and the underlying reference databases, thereby greatly improving our view on 'microbial dark matter'.

Mucisphaera calidilacus gen. nov., sp. nov., a novel planctomycete of the class Phycisphaerae isolated in the shallow sea hydrothermal system of the Lipari Islands.

For extending the current collection of axenic cultures of planctomycetes, we describe in this study the isolation and characterisation of strain Pan265T obtained from a red biofilm in the hydrothermal vent system close to the Lipari Islands in the Tyrrhenian Sea, north of Sicily, Italy. The strain forms light pink colonies on solid medium and grows as a viscous colloid in liquid culture, likely as the result of formation of a dense extracellular matrix observed during electron microscopy. Cells of the novel isolate are spherical, motile and divide by binary fission. Strain Pan265T is mesophilic (temperature optimum 30-33 °C), neutrophilic (pH optimum 7.0-8.0), aerobic and heterotrophic. The strain has a genome size of 3.49 Mb and a DNA G + C content of 63.9%. Phylogenetically, the strain belongs to the family Phycisphaeraceae, order Phycisphaerales, class Phycisphaerae. Our polyphasic analysis supports the delineation of strain Pan265T from the known genera in this family. Therefore, we conclude to assign strain Pan265T to a novel species within a novel genus, for which we propose the name Mucisphaera calidilacus gen. nov., sp. nov. The novel species is the type species of the novel genus and is represented by strain Pan265T (= DSM 100697T = CECT 30425T) as type strain.

Analysis of Bacterial Communities on North Sea Macroalgae and Characterization of the Isolated Planctomycetes Adhaeretor mobilis gen. nov., sp. nov., Roseimaritima multifibrata sp. nov., Rosistilla ulvae sp. nov. and Rubripirellula lacrimiformis sp. nov.

Planctomycetes are bacteria that were long thought to be unculturable, of low abundance, and therefore neglectable in the environment. This view changed in recent years, after it was shown that members of the phylum Planctomycetes can be abundant in many aquatic environments, e.g., in the epiphytic communities on macroalgae surfaces. Here, we analyzed three different macroalgae from the North Sea and show that Planctomycetes is the most abundant bacterial phylum on the alga Fucus sp., while it represents a minor fraction of the surface-associated bacterial community of Ulva sp. and Laminaria sp. Especially dominant within the phylum Planctomycetes were Blastopirellula sp., followed by Rhodopirellula sp., Rubripirellula sp., as well as other Pirellulaceae and Lacipirellulaceae, but also members of the OM190 lineage. Motivated by the observed abundance, we isolated four novel planctomycetal strains to expand the collection of species available as axenic cultures since access to different strains is a prerequisite to investigate the success of planctomycetes in marine environments. The isolated strains constitute four novel species belonging to one novel and three previously described genera in the order Pirellulales, class Planctomycetia, phylum Planctomycetes.

Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation.

As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as "microbial dark matter." In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique-out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages-in whole or in part-have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.

Bremerella alba sp. nov., a novel planctomycete isolated from the surface of the macroalga Fucus spiralis.

A bacterial strain, designated FF15T, was isolated from the thallus surface of the macroalga Fucus spiralis sampled on a rocky beach in Porto, Portugal. Based on the 16S rRNA gene sequence, strain FF15T was affiliated to the phylum Planctomycetes. This strain forms white colonies on modified M13 medium and the cells are pear-shaped, can form rosettes, divide by polar budding and are motile. The novel isolate is mesophilic and neutrophilic with an optimum growth temperature of about 30 °C and an optimum pH for growth between 6.5 and 7.5. It showed growth over a broad range of salinities (0-9% NaCl - optimum at 1.5%). No additional vitamins are required for growth. It is cytochrome c oxidase and catalase positive. The major respiratory quinone was menaquinone 6 (MK-6). Genome sequencing revealed a genome size of 6.37 Mbp and a DNA G + C content of 54.2%. Analysis of phylogenetic markers, including similarities of the 16S rRNA gene sequence, rpoB gene sequence, as well as Percentage of Conserved Proteins (POCP), Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI), suggest the affiliation of strain FF15T to "Bremerella", a recently described genus in the family Pirellulaceae. Based on the genotypic, phylogenetic, chemotaxonomic, physiological and biochemical characterization, we described a new species represented by strain FF15T (=CECT 8078T = LMG 31936T), for which we propose the name Bremerella alba snov.

Analysis of bacterial communities in a municipal duck pond during a phytoplankton bloom and isolation of Anatilimnocola aggregata gen. nov., sp. nov., Lacipirellula limnantheis sp. nov. and Urbifossiella limnaea gen. nov., sp. nov. belonging to the phylum Planctomycetes.

Waterbodies such as lakes and ponds are fragile environments affected by human influences. Suitable conditions can result in massive growth of phototrophs, commonly referred to as phytoplankton blooms. Such events benefit heterotrophic bacteria able to use compounds secreted by phototrophs or their biomass as major nutrient source. One example of such bacteria are Planctomycetes, which are abundant on the surfaces of marine macroscopic phototrophs; however, less data are available on their ecological roles in limnic environments. In this study, we followed a cultivation-independent deep sequencing approach to study the bacterial community composition during a cyanobacterial bloom event in a municipal duck pond. In addition to cyanobacteria, which caused the bloom event, members of the phylum Planctomycetes were significantly enriched in the cyanobacteria-attached fraction compared to the free-living fraction. Separate datasets based on isolated DNA and RNA point towards considerable differences in the abundance and activity of planctomycetal families, indicating different activity peaks of these families during the cyanobacterial bloom. Motivated by the finding that the sampling location harbours untapped bacterial diversity, we included a complementary cultivation-dependent approach and isolated and characterized three novel limnic strains belonging to the phylum Planctomycetes.

Updates to the recently introduced family Lacipirellulaceae in the phylum Planctomycetes: isolation of strains belonging to the novel genera Aeoliella, Botrimarina, Pirellulimonas and Pseudobythopirellula and the novel species Bythopirellula polymerisocia and Posidoniimonas corsicana.

Eight novel strains of the phylum Planctomycetes were isolated from different aquatic habitats. Among these habitats were the hydrothermal vent system close to Panarea Island, a public beach at Mallorca Island, the shore of Costa Brava (Spain), and three sites with brackish water in the Baltic Sea. The genome sizes of the novel strains range from 4.33 to 6.29 Mb with DNA G+C contents between 52.8 and 66.7%. All strains are mesophilic (Topt 24-30 °C) and display generation times between 17 and 94 h. All eight isolates constitute novel species of either already described or novel genera within the family Lacipirellulaceae. Two of the novel species, Posidoniimonas polymericola (type strain Pla123aT = DSM 103020T = LMG 29466T) and Bythopirellula polymerisocia (type strain Pla144T = DSM 104841T = VKM B-3442T), belong to established genera, while the other strains represent the novel genera Aeoliella gen. nov., Botrimarina gen. nov., Pirellulimonas gen. nov. and Pseudobythopirellula gen. nov. Based on our polyphasic analysis, we propose the species Aeoliella mucimassa sp. nov. (type strain Pan181T = DSM 29370T = LMG 31346T = CECT 9840T = VKM B-3426T), Botrimarina colliarenosi sp. nov. (type strain Pla108T = DSM 103355T = LMG 29803T), Botrimarina hoheduenensis sp. nov. (type strain Pla111T = DSM 103485T = STH00945T, Jena Microbial Resource Collection JMRC), Botrimarina mediterranea sp. nov. (type strain Spa11T = DSM 100745T = LMG 31350T = CECT 9852T = VKM B-3431T), Pirellulimonas nuda sp. nov. (type strain Pla175T = DSM 109594T = CECT 9871T = VKM B-3448T) and Pseudobythopirellula maris sp. nov. (type strain Mal64T = DSM 100832T = LMG 29020T).

Additions to the genus Gimesia: description of Gimesia alba sp. nov., Gimesia algae sp. nov., Gimesia aquarii sp. nov., Gimesia aquatilis sp. nov., Gimesia fumaroli sp. nov. and Gimesia panareensis sp. nov., isolated from aquatic habitats of the Northern Hemisphere.

Thirteen novel planctomycetal strains were isolated from five different aquatic sampling locations. These comprise the hydrothermal vent system close to Panarea Island (Italy), a biofilm on the surface of kelp at Monterey Bay (CA, USA), sediment and algae on Mallorca Island (Spain) and Helgoland Island (Germany), as well as a seawater aquarium in Braunschweig, Germany. All strains were shown to belong to the genus Gimesia. Their genomes cover a size range from 7.22 to 8.29 Mb and have a G+C content between 45.1 and 53.7%. All strains are mesophilic (Topt 26-33 °C) with generation times between 12 and 32 h. Analysis of fatty acids yielded palmitic acid (16:0) and a fatty acid with the equivalent chain length of 15.817 as major compounds. While five of the novel strains belong to the already described species Gimesia maris and Gimesia chilikensis, the other strains belong to novel species, for which we propose the names Gimesia alba (type strain Pan241wT = DSM 100744T = LMG 31345T = CECT 9841T = VKM B-3430T), Gimesia algae (type strain Pan161T = CECT 30192T = STH00943T = LMG 29130T), Gimesia aquarii (type strain V144T = DSM 101710T = VKM B-3433T), Gimesia fumaroli (type strain Enr17T = DSM 100710T = VKM B-3429T) and Gimesia panareensis (type strain Enr10T = DSM 100416T = LMG 29082T). STH numbers refer to the Jena Microbial Resource Collection (JMRC).

Author Correction: The planctomycete Stieleria maiorica Mal15T employs stieleriacines to alter the species composition in marine biofilms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: The planctomycete Stieleria maiorica Mal15T employs stieleriacines to alter the species composition in marine biofilms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stieleriacines, N-Acyl Dehydrotyrosines From the Marine Planctomycete Stieleria neptunia sp. nov.

Bacteria of the phylum Planctomycetes occur ubiquitously in marine environments and play important roles in the marine nitrogen- and carbon cycle, for example as scavengers after phototrophic blooms. Here, we describe the isolation and characterization of the planctomycetal strain Enr13T isolated from a Posidonia sp. biofilm obtained from seawater sediment close to Panarea Island, Italy. Phylogenetic tree reconstruction based on 16S rRNA gene sequences and multi-locus sequence analysis supports the delineation of strain Enr13T from characterized species part of the phylum of Planctomycetes. HPLC-MS analysis of culture broth obtained from strain Enr13T revealed the presence of lipophilic metabolites, of which the major compound was isolated by preparative reversed-phase HPLC. The structure of this compound, named stieleriacine D (1), was elucidated utilizing HRESIMS, 1D- and 2D-NMR data as a new N-acylated dehydrotyrosine derivative. Its biosynthesis was proposed based on an in silico gene cluster analysis. Through analysis of the MS/MS spectrum of 1 and its minor derivative, stieleriacine E (2), it was possible to assign the structure of 2 without isolation. 1 showed antibacterial activity, however, the wide distribution of structurally related compounds indicates a potential role as a signaling molecule.

Stieleria varia sp. nov., isolated from wood particles in the Baltic Sea, constitutes a novel species in the family Pirellulaceae within the phylum Planctomycetes.

Species belonging to the bacterial phylum Planctomycetes are ubiquitous members of the microbial communities in aquatic environments and are frequently isolated from various biotic and abiotic surfaces in marine and limnic water bodies. Planctomycetes have large genomes of up to 12.4 Mb, follow complex lifestyles and display an uncommon cell biology; features which motivate the investigation of members of this phylum in greater detail. As a contribution to the current collection of axenic cultures of Planctomycetes, we here describe strain Pla52T isolated from wood particles in the Baltic Sea. Phylogenetic analysis places the strain in the family Pirellulaceae and suggests two species of the recently described genus Stieleria as current closest neighbours. Strain Pla52nT shows typical features of members of the class Planctomycetia, including division by polar budding and the presence of crateriform structures. Colonies of strain Pla52nT have a light orange colour, which is an unusual pigmentation compared to the majority of members in the phylum, which show either a pink to red pigmentation or entirely lack pigmentation. Optimal growth of strain Pla52nT at 33 °C and pH 7.5 indicates a mesophilic (i.e. with optimal growth between 20 and 45 °C) and neutrophilic growth profile. The strain is an aerobic heterotroph with motile daughter cells. Its genome has a size of 9.6 Mb and a G + C content of 56.0%. Polyphasic analyses justify delineation of the strain from described species within the genus Stieleria. Therefore, we conclude that strain Pla52nT = LMG 29463T = VKM B-3447T should be classified as the type strain of a novel species, for which we propose the name Stieleria varia sp. nov.

The Microbiome of Posidonia oceanica Seagrass Leaves Can Be Dominated by Planctomycetes.

Seagrass meadows are ubiquitous, fragile and endangered marine habitats, which serve as fish breeding grounds, stabilize ocean floor substrates, retain nutrients and serve as important carbon sinks, counteracting climate change. In the Mediterranean Sea, seagrass meadows are mostly formed by the slow-growing endemic plant Posidonia oceanica (Neptune grass), which is endangered by global warming and recreational motorboating. Despite its importance, surprisingly little is known about the leaf surface microbiome of P. oceanica. Using amplicon sequencing, we here show that species belonging to the phylum Planctomycetes can dominate the biofilms of young and aged P. oceanica leaves. Application of selective cultivation techniques allowed for the isolation of two novel planctomycetal strains belonging to two yet uncharacterized genera.

Three Planctomycetes isolated from biotic surfaces in the Mediterranean Sea and the Pacific Ocean constitute the novel species Symmachiella dynata gen. nov., sp. nov. and Symmachiella macrocystis sp. nov.

Planctomycetes is a phylum of environmentally important bacteria, which also receive significant attention due to their fascinating cell biology. Access to axenic Planctomycete cultures is crucial to study cell biological features within this phylum in further detail. In this study, we characterise three novel strains, Mal52T, Pan258 and CA54T, which were isolated close to the coasts of the islands Mallorca (Spain) and Panarea (Italy), and from Monterey Bay, CA, USA. The three isolates show optimal growth at temperatures between 22 and 24 °C and at pH 7.5, divide by polar budding, lack pigmentation and form strong aggregates in liquid culture. Analysis of five phylogenetic markers suggests that the strains constitute two novel species within a novel genus in the family Planctomycetaceae. The strains Mal52T (DSM 101177T = VKM B-3432T) and Pan258 were assigned to the species Symmachiella dynata gen nov., sp. nov., while strain CA54T (DSM 104301T = VKM B-3450T) forms a separate species of the same genus, for which we propose the name Symmachiella macrocystis sp. nov.

Rosistilla oblonga gen. nov., sp. nov. and Rosistilla carotiformis sp. nov., isolated from biotic or abiotic surfaces in Northern Germany, Mallorca, Spain and California, USA.

Planctomycetes are ubiquitous bacteria with fascinating cell biological features. Strains available as axenic cultures in most cases have been isolated from aquatic environments and serve as a basis to study planctomycetal cell biology and interactions in further detail. As a contribution to the current collection of axenic cultures, here we characterise three closely related strains, Poly24T, CA51T and Mal33, which were isolated from the Baltic Sea, the Pacific Ocean and the Mediterranean Sea, respectively. The strains display cell biological features typical for related Planctomycetes, such as division by polar budding, presence of crateriform structures and formation of rosettes. Optimal growth was observed at temperatures of 30-33 °C and at pH 7.5, which led to maximal growth rates of 0.065-0.079 h-1, corresponding to generation times of 9-11 h. The genomes of the novel isolates have a size of 7.3-7.5 Mb and a G + C content of 57.7-58.2%. Phylogenetic analyses place the strains in the family Pirellulaceae and suggest that Roseimaritima ulvae and Roseimaritima sediminicola are the current closest relatives. Analysis of five different phylogenetic markers, however, supports the delineation of the strains from members of the genus Roseimaritima and other characterised genera in the family. Supported by morphological and physiological differences, we conclude that the strains belong to the novel genus Rosistilla gen. nov. and constitute two novel species, for which we propose the names Rosistilla carotiformis sp. nov. and Rosistilla oblonga sp. nov. (the type species). The two novel species are represented by the type strains Poly24T (= DSM 102938T = VKM B-3434T = LMG 31347T = CECT 9848T) and CA51T (= DSM 104080T = LMG 29702T), respectively.

Caulifigura coniformis gen. nov., sp. nov., a novel member of the family Planctomycetaceae isolated from a red biofilm sampled in a hydrothermal area.

Pan44T, a novel strain belonging to the phylum Planctomycetes, was isolated from a red biofilm in a hydrothermal area close to the island Panarea in the Tyrrhenian Sea north of Sicily, Italy. The strain forms white colonies on solid medium and displays the following characteristics: cell division by budding, formation of rosettes, presence of matrix or fimbriae and long stalks. The cell surface has an interesting and characteristic texture made up of triangles and rectangles, which leads to a pine cone-like morphology of the strain. Strain Pan44T is mesophilic (temperature optimum 26 °C), slightly alkaliphilic (pH optimum 8.0), aerobic and heterotrophic. The strain has a genome size of 6.76 Mb with a G + C content of 63.2%. Phylogenetically, the strain is a member of the family Planctomycetaceae, order Planctomycetales, class Planctomycetia. Our analysis supports delineation of strain Pan44T from all known genera in this family, hence, we propose to assign it to a novel species within a novel genus, for which we propose the name Caulifigura coniformis gen. nov., sp. nov., represented by Pan44T (DSM 29405T = LMG 29788T) as the type strain.