Genome-resolved metagenomics reveals the effect of nutrient availability on bacterial genomic properties across 44 European freshwater lakes.

Understanding intricate microbial interactions in the environment is crucial. This is especially true for the relationships between nutrients and bacteria, as phosphorus, nitrogen and organic carbon availability are known to influence bacterial population dynamics. It has been suggested that low nutrient conditions prompt the evolutionary process of genome streamlining. This process helps conserve scarce nutrients and allows for proliferation. Genome streamlining is associated with genomic properties such as %GC content, genes encoding sigma factors, percent coding regions, gene redundancy, and functional shifts in processes like cell motility and ATP binding cassette transporters, among others. The current study aims to unveil the impact of nutrition on the genome size, %GC content, and functional properties of pelagic freshwater bacteria. We do this at finer taxonomic resolutions for many metagenomically characterized communities. Our study confirms the interplay of trophic level and genomic properties. It also highlights that different nutrient types, particularly phosphorus and nitrogen, impact these properties differently. We observed a covariation of functional traits with genome size. Larger genomes exhibit enriched pathways for motility, environmental interaction, and regulatory genes. ABC transporter genes reflect the availability of nutrients in the environment, with small genomes presumably relying more on metabolites from other organisms. We also discuss the distinct strategies different phyla adopt to adapt to oligotrophic environments. The findings contribute to our understanding of genomic adaptations within complex microbial communities.

Description of a new freshwater bacterium Aquirufa regiilacus sp. nov., classification of the genera Aquirufa, Arundinibacter, Sandaracinomonas, and Tellurirhabdus to the family Spirosomataceae, classification of the genus Chryseotalea to the family Fulvivirgaceae and Litoribacter to the family Cyclobacteriaceae, as well as classification of Litoribacter alkaliphilus as a later heterotypic synonym of Litoribacter ruber.

Strains LEOWEIH-7CT and LEPPI-3A were isolated from the Leopoldskroner Weiher, a lake located in the city of Salzburg, Austria. 16S rRNA gene similarities and phylogenetic reconstructions with 16S rRNA gene sequences as well as based on genome sequences revealed that the new strains belong to the A. antheringensis branch of the genus Aquirufa. Calculated whole-genome average nucleotide identity (gANI) and digital DNA-DNA hybridization (dDDH) values with the closely related type strains showed that the two strains represent a single new species. The strains grew aerobically and chemoorganotrophically, and the cells were rod shaped, on average 0.8 µm long and 0.3 µm wide, red pigmented and motile by gliding. The genome size of both strains was 2.6 Mbp and the G+C value was 41.9%. The genomes comprised genes predicted for the complete light-harvesting rhodopsin system and various carotenoids. We proposed to establish the name Aquirufa regiilacus sp. nov. for strain LEOWEIH-7CT (=DSM 116390T = JCM 36347T) as the type strain. Strain LEPPI-3A (=DSM 116391 = JCM 36348) also belongs to this new species. The calculated genome-based phylogenetic tree revealed that Aquirufa and some other genera currently allocated in the family Cytophagaceae need a reclassification. Aquirufa, Arundinibacter, Sandaracinomonas, and Tellurirhabdus should be designated to the family Spirosomataceae, the genus Chryseotalea to the family Fulvivirgaceae, and the genus Litoribacter to the family Cyclobacteriaceae. Furthermore, based on calculated gANI and dDDH values, Litoribacter alkaliphilus should be reclassified as a later heterotypic synonym of Litoribacter ruber.

Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens from Alpine Areas in Eastern Alps and Equatorial Africa.

The diversity of bacteria associated with alpine lichens was profiled. Lichen samples belonging to the Umbilicariaceae family, commonly known as rock tripe lichens, were gathered from two distinct alpine fellfields: one situated on Mt. Brennkogel located in the Eastern European Alps (Austria), and the other on Mt. Stanley located in the Rwenzori mountains of equatorial Africa (Uganda). The primary aim of this research was to undertake a comparative investigation into the bacterial compositions, and diversities, identifying potential indicators and exploring their potential metabolisms, of these lichen samples. Bulk genomic DNA was extracted from the lichen samples, which was used to amplify the 18S rRNA gene by Sanger sequencing and the V3-V4 region of the 16S rRNA gene by Illumina Miseq sequencing. Examination of the fungal partner was carried out through the analysis of 18S rRNA gene sequences, belonging to the genus Umbilicaria (Ascomycota), and the algal partner affiliated with the lineage Trebouxia (Chlorophyta), constituted the symbiotic components. Analyzing the MiSeq datasets by using bioinformatics methods, operational taxonomic units (OTUs) were established based on a predetermined similarity threshold for the V3-V4 sequences, which were assigned to a total of 26 bacterial phyla that were found in both areas. Eight of the 26 phyla, i.e. Acidobacteriota, Actinomycota, Armatimonadota, Bacteroidota, Chloroflexota, Deinococcota, Planctomycetota, and Pseudomonadota, were consistently present in all samples, each accounting for more than 1% of the total read count. Distinct differences in bacterial composition emerged between lichen samples from Austria and Uganda, with the OTU frequency-based regional indicator phyla, Pseudomonadota and Armatimonadota, respectively. Despite the considerable geographic separation of approximately 5430 km between the two regions, the prediction of potential metabolic pathways based on OTU analysis revealed similar relative abundances. This similarity is possibly influenced by comparable alpine climatic conditions prevailing in both areas.

Aquirufa lenticrescens sp. nov. and Aquirufa aurantiipilula sp. nov.: two new species of a lineage of widespread freshwater bacteria.

Two bacterial strains, 9H-EGSET and 15D-MOBT, were isolated from small freshwater habitats located near Salzburg, Austria. They showed the highest 16S rRNA sequence similarities of 100% and 99.9%, respectively, with type strains of species of the genus Aquirufa (Bacteroidota). Genome-based phylogenetic reconstructions with 119 amino acid sequences assigned the new taxa to the two distinct branches of the genus Aquirufa. Whole-genome average nucleotide identities were calculated with all possible pairs belonging to the genus. Values between 75.4% and 88.6% revealed that the two new strains represent each a new species. Like all, so far described members of the genus, they grew aerobically and chemoorganotrophically, were rod-shaped, red-pigmented, and motile by gliding, and showed genome sizes of about 3 Mbp and G + C values of about 40%. They could be distinguished by some phenotypic and chemotaxonomic features from their nearest related species. Until now, strain 9H-EGSET is the only one among the Aquirufa strains which contained traces of MK8 as respiratory quinone, and strain 15D-MOBT is the only one that formed tiny orange globules in liquid medium. The genome of strain 9H-EGSET comprised genes for the complete light-harvesting rhodopsin / retinal system, in the case of 15D-MOBT genes predicted for a nitrous oxide reductase were present. For the two new species of the genus Aquirufa, we propose to establish the names Aquirufa lenticrescens for strain 9H-EGSET (= JCM 34077 T = CIP 111926 T) and Aquirufa aurantiipilula for strain 15D-MOBT (= JCM 34078 T = CIP 111925 T).

Fourteen new Polynucleobacter species: P. brandtiae sp. nov., P. kasalickyi sp. nov., P. antarcticus sp. nov., P. arcticus sp. nov., P. tropicus sp. nov., P. bastaniensis sp. nov., P. corsicus sp. nov., P. finlandensis sp. nov., P. ibericus sp. nov., P. hallstattensis sp. nov., P. alcilacus sp. nov., P. nymphae sp. nov., P. paludilacus sp. nov. and P. parvulilacunae sp. nov.

Fourteen strains, all isolated from the surface of freshwater habitats, were genomically, phylogenetically and phenotypically characterized. The strains were obtained from geographically and climatically broadly scattered sites. This included two lakes in Antarctica, one arctic pond located on the Svalbard archipelago (Norway), a tropical habitat located in Uganda, some lakes in Southern Europe (Spain and France), lakes, ponds and a puddle in Central Europe (Austria, Czech Republic and Germany), and lakes in Northern Europe (Finland). Most of the investigated strains were characterized by rather small cell sizes and rather slow growth on media such as nutrient broth-soyotone-yeast extract (NSY) medium. Phylogenomic analyses indicated that all fourteen strains are affiliated with the genus Polynucleobacter (Burkholderiaceae, Pseudomonadota). Thirteen of the strains were found to be affiliated with subcluster PnecC of the genus. All these strains were characterized by genome sizes in the range of 1.7-2.3 Mbp and G+C values of 44.9-46.5 mol%. Furthermore, all PnecC-affiliated strains shared 16S rRNA gene sequence similarities >99 %. Only one strain characterized by a larger genome size of 2.9 Mbp and a lower G+C value of 41.0 mol% was found to be affiliated with subcluster PnecA. Whole genome sequence comparisons revealed that all 14 strains shared among each other, as well as with the type strains of the previously described 17 Polynucleobacter species, whole-genome average nucleotide identities values <95 %. This suggested that the 14 investigated strains represent 14 different new species. We propose the establishment of 14 new Polynucleobacter species represented by the following type strains: UB-Domo-W1T (=DSM 103491T=CIP 111598T=JCM 32562T), VK13T (=DSM 103488T=JCM 32564T), LimPoW16T (=DSM 24085T=CIP 111098T), UK-Long2-W17T (=DSM 103489T=CIP 111328T=JCM 32563T), UK-Pondora-W15T (=DSM 103423T=JCM 32939T), MWH-Mekk-B1T (=DSM 106779T=JCM 32556T), MWH-Mekk-C3T (=DSM 103415T=JCM 32557T), Ross1-W9T (=DSM 103416T=JCM 32561T), MWH-Hall10T (=DSM 107042T=JCM 32938T), AP-Basta-1000A-D1T (=DSM 107039T=JCM 32933T), AP-Melu-1000-A1T (=DSM 107036T=JCM 32935T), es-MAR-2T (=DSM 103424T=JCM 32554T), AP-Mumm-500A-B3T (=DSM 107037T=JCM 32936T), MWH-UH21BT (=DSM 23884T=LMG 29707T).

Zwartia hollandica gen. nov., sp. nov., Jezberella montanilacus gen. nov., sp. nov. and Sheuella amnicola gen. nov., comb. nov., representing the environmental GKS98 (betIII) cluster.

We present two strains affiliated with the GKS98 cluster. This phylogenetically defined cluster is representing abundant, mainly uncultured freshwater bacteria, which were observed by many cultivation-independent studies on the diversity of bacteria in various freshwater lakes and streams. Bacteria affiliated with the GKS98 cluster were detected by cultivation-independent methods in freshwater systems located in Europe, Asia, Africa and the Americas. The two strains, LF4-65T (=CCUG 56422T=DSM 107630T) and MWH-P2sevCIIIbT (=CCUG 56420T=DSM 107629T), are aerobic chemoorganotrophs, both with genome sizes of 3.2 Mbp and G+C values of 52.4 and 51.0 mol%, respectively. Phylogenomic analyses based on concatenated amino acid sequences of 120 proteins suggest an affiliation of the two strains with the family Alcaligenaceae and revealed Orrella amnicola and Orrella marina (= Algicoccus marinus) as being the closest related, previously described species. However, the calculated phylogenomic trees clearly suggest that the current genus Orrella represents a polyphyletic taxon. Based on the branching order in the phylogenomic trees, as well as the revealed phylogenetic distances and chemotaxonomic traits, we propose to establish the new genus Zwartia gen. nov. and the new species Z. hollandica sp. nov. to harbour strain LF4-65T and the new genus Jezberella gen. nov. and the new species J. montanilacus sp. nov. to harbour strain MWH-P2sevCIIIbT. Furthermore, we propose the reclassification of the species Orrella amnicola in the new genus Sheuella gen. nov. The new genera Zwartia, Jezberella and Sheuella together represent taxonomically the GKS98 cluster.

Aquiluna borgnonia gen. nov., sp. nov., a member of a Microbacteriaceae lineage of freshwater bacteria with small genome sizes.

The actinobacterial strain 15G-AUS-rotT was isolated from an artificial pond located near Salzburg, Austria. The strain showed 16S rRNA gene sequence similarities of 98.7 % to Candidatus Aquiluna rubra and of 96.6 and 96.7 % to the two validly described species of the genus Rhodoluna. Phylogenetic reconstructions based on 16S rRNA gene sequences and genome-based on amino acid sequences of 118 single copy genes referred strain 15G-AUS-rotT to the family Microbacteriaceae and therein to the so-called subcluster Luna-1. The genome-based phylogenetic tree showed that the new strain represents a putative new genus. Cultures of strain 15G-AUS-rotT were light red pigmented and comprised very small, rod-shaped cells. They metabolized a broad variety of substrates. Major fatty acids (>10 %) of cells were iso-C16 : 0, antiso-C15 : 0 and iso-C14 : 0. The major respiratory quinone was MK-11 and a minor component was MK-10. The peptidoglycan structure belonged to an unusual B type. The closed genome sequence of the strain was very small (1.4 Mbp) and had a DNA G+C content of 54.8 mol%. An interesting feature was the presence of genes putatively encoding the complete light-driven proton pumping actinorhodopsin/retinal system, which were located at three different positions of the genome. Based on the characteristics of the strain, a new genus and a new species termed Aquiluna borgnonia is proposed for strain 15G-AUS-rotT (=DSM 107803T=JCM 32974T).

Aurantimicrobium photophilum sp. nov., a non-photosynthetic bacterium adjusting its metabolism to the diurnal light cycle and reclassification of Cryobacterium mesophilum as Terrimesophilobacter mesophilus gen. nov., comb. nov.

The aerobic primarily chemoorganotrophic actinobacterial strain MWH-Mo1T was isolated from a freshwater lake and is characterized by small cell lengths of less than 1 µm, small cell volumes of 0.05-0.06 µm3 (ultramicrobacterium), a small genome size of 1.75 Mbp and, at least for an actinobacterium, a low DNA G+C content of 54.6 mol%. Phylogenetic analyses based on concatenated amino acid sequences of 116 housekeeping genes suggested the type strain of Aurantimicrobium minutum affiliated with the family Microbacteriaceae as its closest described relative. Strain MWH-Mo1T shares with the type strain of that species a 16S rRNA gene sequence similarity of 99.6 % but the genomes of the two strains share an average nucleotide identity of only 79.3 %. Strain MWH-Mo1T is in many genomic, phenotypic and chemotaxonomic characteristics quite similar to the type strain of A. minutum. Previous intensive investigations revealed two unusual traits of strain MWH-Mo1T. Although the strain is not known to be phototrophic, the metabolism is adjusted to the diurnal light cycle by up- and down-regulation of genes in light and darkness. This results in faster growth in the presence of light. Additionally, a cell size-independent protection against predation by bacterivorous flagellates, most likely mediated by a proteinaceous cell surface structure, was demonstrated. For the previously intensively investigated aerobic chemoorganotrophic actinobacterial strain MWH-Mo1T (=CCUG 56426T=DSM 107758T), the establishment of the new species Aurantimicrobium photophilum sp. nov. is proposed.

Aquirufa ecclesiirivi sp. nov. and Aquirufa beregesia sp. nov., isolated from a small creek and classification of Allopseudarcicella aquatilis as a later heterotypic synonym of Aquirufa nivalisilvae.

Two bacterial strains, 50A-KIRBAT and 50C-KIRBAT, were isolated from the same freshwater creek located near Salzburg, Austria. They showed 16S rRNA gene sequence similarities to Aquirufa nivalisilvae of 100 and 99.9 %, respectively. A genome-based phylogenetic reconstruction with amino acid sequences of 119 single-copy genes suggested that the new strains represent two new species of the genus Aquirufa. Pairwise calculated whole-genome average nucleotide identity (gANI) values ranging from 85.4 to 87.5 % confirmed this conclusion. Phenotypic, chemotaxonomic and genomic traits were investigated. Like strains of other Aquirufa species, 50A-KIRBAT and 50C-KIRBAT grew aerobically and chemoorganotrophically, were rod-shaped, red-pigmented and motile, most likely by gliding. They could be distinguished by slight differences in the chemotaxonomic features. We propose to establish for strain 50A-KIRBAT (=CIP 111735T=LMG 31080T) as type strain the name Aquirufa ecclesiirivi and for strain 50C-KIRBAT (=CIP 111736T=LMG 31501T) as type strain the name Aquirufa beregesia. Furthermore, the relationship between the type strains of Aquirufa nivalisilvae (59G-WUEMPELT) and Allopseudarcicella aquatilis (HME7025T) was investigated. Results of polyphasic analyses, especially a gANI value of 97.6 %, as well as the genome-based phylogenetic reconstruction, suggested that Allopseudarcicella aquatilis is a heterotypic synonym of Aquirufa nivalisilvae. According to rule 24b of the International Code of Nomenclature of Prokaryotes we propose to classify strain HME7025 as Aquirufa nivalisilvae and provide an emended description for the latter.

Fluviispira multicolorata gen. nov., sp. nov. and Silvanigrella paludirubra sp. nov., isolated from freshwater habitats.

Strain 33A1-SZDPT was isolated from a small creek located in Puch, Austria. Strain SP-Ram-0.45-NSY-1T was obtained from a small pond located in Schönramer Moor, Germany. 16S rRNA gene sequence similarities between the type strain of Silvanigrella aquatica, currently the only member of the family Silvanigrellaceae, and strains 33A1-SZDPT and SP-Ram-0.45-NSY-1T of 94.1 and 99.1 %, respectively, suggested affiliation of the two strains with this family. Phylogenetic reconstructions with 16S rRNA gene sequences and phylogenomic analyses with amino acid sequences obtained from 103 single-copy genes suggested that the strains represent a new genus and a new species in the case of strain 33A1-SZDPT (=JCM 32978T=DSM 107810T), and a new species within the genus Silvanigrella in the case of strain SP-Ram-0.45-NSY-1T (=JCM 32975T=DSM 107809T). Cells of strain 33A1-SZDPT were motile, pleomorphic, purple-pigmented on agar plates, putatively due to violacein, and showed variable pigmentation in liquid media. They grew chemoorganotrophically and aerobically and tolerated salt concentrations up to 1.2 % NaCl (v/w). The genome size of strain 33A1-SZDPT was 3.4 Mbp and the G+C content was 32.2 mol%. For this new genus and new species, we propose the name Fluviispira multicolorata gen. nov., sp. nov. Cells of strain SP-Ram-0.45-NSY-1T were motile, pleomorphic, red-pigmented and grew chemoorganotrophically and aerobically. They tolerated salt concentrations up to 1.1 % NaCl (v/w). The genome size of strain SP-Ram-0.45-NSY-1T was 3.9 Mbp and the G+C content 29.3 mol%. For the new species within the genus Silvanigrella we propose the name Silvanigrella paludirubra sp. nov.

Rariglobus hedericola gen. nov., sp. nov., belonging to the Verrucomicrobia, isolated from a temperate freshwater habitat.

The bacterial strain 53C-WASEF was isolated from a small freshwater ditch located in Eugendorf, Austria. Phylogenetic reconstructions with 16S rRNA gene sequences and genome based, with amino acid sequences obtained from 105 single copy genes, suggested that the strain represents a new genus and a new species within the family Opitutaceae, which belongs to the class Opitutae of the phylum Verrucomicrobia. Comparisons of the 16S rRNA gene sequence of strain 53C-WASEF with those of related type strains revealed a highest sequence similarity of 93.5 % to Nibricoccus aquaticus and of 92.9 % to Geminisphaera colitermitum. Interestingly, phylogentic trees indicated the latter as being the closest known relative of the new strain. Phenotypic, chemotaxonomic and genomic traits were investigated. Cells were observed to be small, spherical, motile and unpigmented, and grew chemoorganotrophically and aerobically. The respiratory quinone was MK-7, the predominant fatty acids were anteiso-C15 : 0, C16 : 1ω5c and C16 : 0. The identified polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Genome sequencing revealed genes putatively encoding for flagella synthesis and cellulose degradation. The genome size was 4.1 Mbp and the G+C content 60.6 mol%. For the new genus and the new species, we propose the name Rariglobus hedericola gen. nov., sp. nov. (=CIP 111665T=DSM 109123T).

Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities.

The class Deltaproteobacteria comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum Proteobacteria, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class Deltaproteobacteria encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the Oligoflexia. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes Deltaproteobacteria and Oligoflexia in the phylum Proteobacteria. Instead, the great majority of currently recognized members of the class Deltaproteobacteria are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class Oligoflexia represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum Thermodesulfobacteria, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the Thermodesulfobacteria rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.

Light Modulates the Physiology of Nonphototrophic Actinobacteria.

Light is a source of energy and an environmental cue that is available in excess in most surface environments. In prokaryotic systems, conversion of light to energy by photoautotrophs and photoheterotrophs is well understood, but the conversion of light to information and the cellular response to that information have been characterized in only a few species. Our goal was to explore the response of freshwater Actinobacteria, which are ubiquitous in illuminated aquatic environments, to light. We found that Actinobacteria without functional photosystems grow faster in the light, likely because sugar transport and metabolism are upregulated in the light. Based on the action spectrum of the growth effect and comparisons of the genomes of three Actinobacteria with this growth rate phenotype, we propose that the photosensor in these strains is a putative CryB-type cryptochrome. The ability to sense light and upregulate carbohydrate transport during the day could allow these cells to coordinate their time of maximum organic carbon uptake with the time of maximum organic carbon release by primary producers.IMPORTANCE Sunlight provides information about both place and time. In sunlit aquatic environments, primary producers release organic carbon and nitrogen along with other growth factors during the day. The ability of Actinobacteria to coordinate organic carbon uptake and utilization with production of photosynthate enables them to grow more efficiently in the daytime, and it potentially gives them a competitive advantage over heterotrophs that constitutively produce carbohydrate transporters, which is energetically costly, or produce transporters only after detection of the substrate(s), which delays their response. Understanding how light cues the transport of organic carbon and its conversion to biomass is key to understanding biochemical mechanisms within the carbon cycle, the fluxes through it, and the variety of mechanisms by which light enhances growth.

Spatial insurance in multi-trophic metacommunities.

Metacommunity theory suggests that dispersal is a key driver of diversity and ecosystem functioning in changing environments. The capacity of dispersal to mitigate effects of environmental change might vary among trophic groups, potentially resulting in changes in trophic interactions and food web structure. In a mesocosm experiment, we compared the compositional response of bacteria, phyto- and zooplankton to a factorial manipulation of acidification and dispersal. We found that the buffering capacity of dispersal varied among trophic groups: dispersal alleviated the negative effect of acidification on phytoplankton diversity mid-experiment, but had no effect on the diversity of zooplankton and bacteria. Likewise, trophic groups differed in whether dispersal facilitated compositional change. Dispersal accelerated changes in phytoplankton composition under acidification, possibly mediated by changes in trophic interactions, but had no effect on the composition of zooplankton and bacteria. Overall, our results suggest that the potential for spatial insurance can vary among trophic groups.

Rhodoluna limnophila sp. nov., a bacterium with 1.4 Mbp genome size isolated from freshwater habitats located in Salzburg, Austria.

Three actinobacterial strains, 27D-LEPIT, 1B-Mac and 36A-HELLB, were isolated from small standing and running freshwater habitats located in Salzburg, Austria. Phylogenetic reconstructions based on 16S rRNA gene sequences and genome based on concatenated amino acid sequences of 119 single-copy genes referred the three strains within the family Microbacteriaceae to the genus Rhodoluna. The strains showed 100 % 16S rRNA gene sequence similarities to the previously described Candidatus Rhodoluna limnophila. Cells were very small, approximately 0.5×0.3 µm, and showed a red pigmentation in liquid nutrient broth-soyotone-yeast extract medium as well as on agar plates. The strains assimilated a broad variety of substrates; however, the patterns differed a great deal between the three investigated strains. For strain 27D-LEPIT, the major fatty acids were iso-C14 : 0 and anteiso-C15 : 0; the identified polar lipids were phosphatidylglycerol and diphosphatidylglycerol; the major respiratory quinone was MK-11; and analysis of the peptidoglycan structure indicated the presence of type B2ß (typeB10). The genomic DNA of the three strains was very small (1.4 Mbp) and had a DNA G+C content of 54 mol%. The investigated traits suggested that strains 36A-HELLB (=DSM 107805=JCM 32927), 1B-Mac (=DSM 107802=JCM 32925) and 27D-LEPIT (=JCM 32926T =DSM 107804T) represent a new species for which the name Rhodolunalimnophila sp. nov. is proposed, with strain 27D-LEPIT as the type strain.

Aquirufa antheringensis gen. nov., sp. nov. and Aquirufa nivalisilvae sp. nov., representing a new genus of widespread freshwater bacteria.

Three bacterial strains, 30S-ANTBAC, 103A-SOEBACH and 59G- WUEMPEL, were isolated from two small freshwater creeks and an intermittent pond near Salzburg, Austria. Phylogenetic reconstructions with 16S rRNA gene sequences and, genome based, with amino acid sequences obtained from 119 single copy genes showed that the three strains represent a new genus of the family Cytophagaceae within a clade formed by the genera Pseudarcicella, Arcicella and Flectobacillus. blast searches suggested that the new genus comprises widespread freshwater bacteria. Phenotypic, chemotaxonomic and genomic traits were investigated. Cells were rod shaped and were able to glide on soft agar. All strains grew chemoorganotrophically and aerobically, were able to assimilate pectin and showed an intense red pigmentation putatively due to various carotenoids. Two strains possessed genes putatively encoding proteorhodopsin and retinal biosynthesis. Genome sequencing revealed genome sizes between 2.5 and 3.1 Mbp and G+C contents between 38.0 and 42.7 mol%. For the new genus we propose the name Aquirufa gen. nov. Pairwise-determined whole-genome average nucleotide identity values suggested that the three strains represent two new species within the new genus for which we propose the names Aquirufa antheringensis sp. nov. for strain 30S-ANTBACT (=JCM 32977T =LMG 31079T=DSM 108553T) as type species of the genus, to which also belongs strain 103A-SOEBACH (=DSM 108555=LMG 31082) and Aquirufa nivalisilvae sp. nov. for strain 59G-WUEMPELT (=LMG 31081T =DSM 108554T).

Polynucleobacter paneuropaeus sp. nov., characterized by six strains isolated from freshwater lakes located along a 3000 km north-south cross-section across Europe.

Six Polynucleobacter (Burkholderiaceae, Betaproteobacteria) strains isolated from different freshwater lakes located across Europe were taxonomically investigated. Phylogenetic analyses based on 16S rRNA gene sequences assigns all six strains to the cryptic species complex PnecC within the genus Polynucleobacter. Analyses of partial glutamine synthetase (glnA) genes suggests that all six strains belong to the species-like taxon designated F15 in previous papers. Comparative genome analyses reveal that the six strains form a genomically coherent group characterized by whole-genome average nucleotide identity (gANI) values of >98 % but separated by gANI values of <88 % from the type strains and representatives of the 16 previously described Polynucleobacter species. In phylogenetic analyses based on nucleotide sequences of 319 orthologous genes, the six strains represent a monophyletic cluster that is clearly separated from all other described species. Genome sizes of the six strains range from 1.61 to 1.83 Mbp, which is smaller than genome sizes of the majority of type strains representing previously described Polynucleobacter species. By contrast, the G+C content of the DNA of the strains is well in the range of 44.8-46.6 mol% previously found for other type strains of species affiliated with the subgroup PnecC. Variation among the six strains representing the new species is evident in a number of traits. These include gene content differences, for instance regarding a gene cluster encoding anoxygenic photosynthesis, as well as phenotypic traits. We propose to name the new species represented by the six strains Polynucleobacter paneuropaeus sp. nov. and designate strain MG-25-Pas1-D2T (=DSM 103454T =CIP 111323T) as the type strain.

Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov., both isolated from freshwater systems.

Strains MWH-EgelM1-30-B4T and MWH-Feld-100T were isolated from the water columns of two freshwater systems. Both strains represent delicate bacteria not easy to work with in laboratory experiments. Phylogenetic analyses of the 16S rRNA genes suggested that both strains were affiliated with the genus Polynucleobacter. Both strains share 16S rRNA gene sequence similarities of >99 % with eight free-living Polynucleobacter type strains, all affiliated with the cryptic species complex PnecC. The full-length 16S rRNA gene sequences of the two strains differ only in two and three positions, respectively, from the sequence of the closest related Polynucleobacter type strain. Genome sequencing of both strains revealed relatively small genome sizes of 2.0 Mbp and G+C contents of 45 mol%. Phylogenetic analyses based on nucleotide sequences of 319 shared protein-encoding genes consistently placed the two strains in taxon PnecC but did not suggest an affiliation with one of the previously described species. Pairwise analyses of whole genome average nucleotide identities (gANI) with representatives of all previously described Polynucleobacter species resulted in both cases throughout in values <80 %. Pairwise comparison of the genomes of the two new strains resulted in gANI values of 83.3 %. All gANI analyses clearly suggested that strains MWH-EgelM1-30-B4T and MWH-Feld-100T represent two novel Polynucleobacter species. We propose for these novel species the names Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov. and strains MWH-EgelM1-30-B4T (=DSM 23911T=LMG 30144T) and MWH-Feld-100T (=DSM 24007T=LMG 29705T) as the type strains, respectively.

Polynucleobacter meluiroseus sp. nov., a bacterium isolated from a lake located in the mountains of the Mediterranean island of Corsica.

Strain AP-Melu-1000-B4 was isolated from a lake located in the mountains of the Mediterranean island of Corsica (France). Phenotypic, chemotaxonomic and genomic traits were investigated. Phylogenetic analyses based on 16S rRNA gene sequencing referred the strain to the cryptic species complex PnecC within the genus Polynucleobacter. The strain encoded genes for biosynthesis of proteorhodopsin and retinal. When pelleted by centrifugation the strain showed an intense rose colouring. Major fatty acids were C16 : 1ω7c, C16 : 0, C18 : 1ω7c and summed feature 2 (C16 : 1 isoI and C14 : 0-3OH). The sequence of the 16S rRNA gene contained an indel which was not present in any previously described Polynucleobacter species. Genome sequencing revealed a genome size of 1.89 Mbp and a G+C content of 46.6 mol%. In order to resolve the phylogenetic position of the new strain within subcluster PnecC, its phylogeny was reconstructed from sequences of 319 shared genes. To represent all currently described Polynucleobacter species by whole genome sequences, three type strains were additionally sequenced. Our phylogenetic analysis revealed that strain AP-Melu-100-B4 occupied a basal position compared with previously described PnecC strains. Pairwise determined whole genome average nucleotide identity (gANI) values suggested that strain AP-Melu-1000-B4 represents a new species, for which we propose the name Polynucleobacter meluiroseus sp. nov. with the type strain AP-Melu-1000-B4T (=DSM 103591T=CIP 111329T).

Photochemical characterization of actinorhodopsin and its functional existence in the natural host.

Actinorhodopsin (ActR) is a light-driven outward H+ pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed λmax at 534nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P620, could be assigned to the O intermediate in other H+ pumps. In contrast to conventional O, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P620, while the other first releases H+ prior to H+ uptake during P620 formation.