Planktophila gen. nov.

The order Nanopelagicales was proposed as Candidatus order by Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). It contains the family Nanopelagicaceae and two genera, Nanopelagicus and Planktophila, type strain for the order is Nanopelagicus abundans MMS-IIB-91 (GCF_002288305.1). Basis of the assignment is a phylogenetic tree of 48 conserved concatenated proteins of >100 complete genomes of all orders of Actinobacteria in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). Aerobic chemoheterotrophs. Cells are tiny, non-motile, and inhabit the plankton of freshwaters. The order is also known as acI or hgc1 (SILVA classification) from 16S rRNA based studies and is one of the most abundant microbes in freshwater lakes. Nanopelagicaceae can be recognized by the presence of the diagnostic oligonucleotide sequence 5’-AATGCGTTAGCTGCGTCGCA-3’ in the 16S rRNA gene (positions 852-872, E. coli numbering). The initial pure cultures were lost after a few propagations to fresh medium, they were isolated in sterile lake water amended with minimal carbon medium, vitamins and amino acids, no growth was observed in rich medium or on agar plates. No growing culture of Nanopelagicus sp. is available, while some Planktophila sp. strains are actively growing in autoclaved lake water (Kim et al. 2019, ISMEJ, doi: 10.1038/s41396-019-0432-x) or artificial media (Salcher et al., in review). None of the isolated strains were yet submitted to a culture collection because these bacteria are hard to maintain, i.e., they are very slowly growing, reach low densities in liquid culture, and do not grow on agar plates.

The family Nanopelagicaceae and the order Nanopelagicales were proposed as Candidatus taxa by Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). It contains two genera, Nanopelagicus and Planktophila, type strain for the family is Nanopelagicus abundans MMS-IIB-91 (GCF_002288305.1). Basis of the assignment is a phylogenetic tree of 48 conserved concatenated proteins of >100 complete genomes of all orders of Actinobacteria in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). Aerobic chemoheterotrophs. Cells are tiny, non-motile, and inhabit the plankton of freshwaters. The family is also known as acI or hgc1 (SILVA classification) from 16S rRNA based studies and is one of the most abundant microbes in freshwater lakes. Nanopelagicaceae can be recognized by the presence of the diagnostic oligonucleotide sequence 5’-AATGCGTTAGCTGCGTCGCA-3’ in the 16S rRNA gene (positions 852-872, E. coli numbering). The initial pure cultures were lost after a few propagations to fresh medium; no growing culture is available. The initial pure cultures were lost after a few propagations to fresh medium, they were isolated in sterile lake water amended with minimal carbon medium, vitamins and amino acids, no growth was observed in rich medium or on agar plates. No growing culture of Nanopelagicus sp. is available, while some Planktophila sp. strains are actively growing in autoclaved lake water (Kim et al. 2019, ISMEJ, doi: 10.1038/s41396-019-0432-x) or artificial media (Salcher et al., in review). None of the isolated strains were yet submitted to a culture collection because these bacteria are hard to maintain, i.e., they are very slowly growing, reach low densities in liquid culture, and do not grow on agar plates.

The genus Nanopelagicus as well as the family Nanopelagicaceae and order Nanopelagicales was proposed as Candidatus by Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). Three strains were isolated from Lake Zurich, Switzerland, that were proposed as Nanopelagicus abundans, Nanopelagicus limnes and Nanopelagicus hibericus, type strain for the genus is Nanopelagicus abundans MMS-IIB-91 (GCF_002288305.1). Basis of the assignment is a phylogenetic tree of 48 conserved concatenated proteins of >100 complete genomes of all orders of Actinobacteria in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivated relatives are Planktophila sp. with average amino acid identities of 62-63.9% and average nucleotide identities of 65.9-68.1%. Aerobic chemoheterotrophs. Cells are tiny, non-motile, and inhabit the plankton of freshwaters. The genus is also known as acI-B1 or hgc1 (SILVA classification) from 16S rRNA based studies and is one of the most abundant genera in freshwater lakes. Members of the genus Nanopelagicus can be recognized by the presence of the diagnostic oligonucleotide sequence 5’-ACAAGAGGTTCGTCCGTCC-3’ in the 23S rRNA gene (positions 2669-2688, E. coli numbering). The initial pure cultures were lost after a few propagations to fresh medium; no growing culture is available. The initial cultures grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. 

The name Planktophila was initially proposed in Jezbera et al. 2009 based on a enrichment culture, with 'Ca. Planktophila limnetica' strain MWH-EgelM2-3.acI as type strain. Initial description: Represented by the aerobic strain MWH-EgelM2-3.acI, which was isolated from a small lake near Salzburg, Austria. Curved rods with lengths of 1.0–1.5 mm and diameters of 0.4–0.5 mm. No pure culture is available. Contained in a stable mixed culture together with ‘Candidatus Limnoluna rubra’ and a Polynucleobacter necessarius subsp. asymbioticus strain. The mixed culture grows well in liquid NSY medium. Supplementation of NSY medium with L-alanine results in an increase of the relative proportion of ‘Ca. Planktophila limnetica’ by more than one order of
magnitude. ‘Ca. Planktophila limnetica’ can be recognized by the presence of the diagnostic oligonucleotide sequence 5'-GAAACTTGGTGGCATCRCCG-3' in the 16S rRNA gene (positions 183–193, E. coli numbering). Phylogenetic analysis of the 16S rRNA gene sequence indicated the affiliation of the Candidatus taxon with the order Actinomycetales.
The genus description was later amended with the first genome-sequenced strains (Neuenschwander et al. 2018) and strain Planktophila limnetica MMS-VB-114 (GCF_002288365.1) as type strain.

Type genome is Nanopelagicus abundans MMS-IIB-91 (GCF_002288305.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.46±0.47 μm and diameters of 0.26±0.20 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Nanopelagicus abundans MMS-IIB-91 has a genome size of 1.16 Mbp and a genomic GC content of 40.2%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 39 tRNAs. It is auxotrophic for reduced sulfur sources, several amino acids (methionine, lysine, ornithine, histidine, betaine) and several vitamins (B1, B5, B7, B12) and possesses rhodopsins. Members of the genus Nanopelagicus can be recognized by the presence of the diagnostic oligonucleotide sequence 5’-ACAAGAGGTTCGTCCGTCC-3’ in the 23S rRNA gene (positions 2669-2688, E. coli numbering). Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Nanopelagicus limnes MMS-21-122 (GCF_002287885.2) and Nanopelagicus hibericus MMS-21-160 (GCF_002288005.1) with average amino acid identities of 81 and 80.7%, respectively, and average nucleotide identities of 77.1 and 76.2%, respectively.

Type genome is Nanopelagicus hibericus MMS-21-160 (GCF_002288005.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.33±0.07 μm and diameters of 0.24±0.03 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Nanopelagicus hibericus MMS-21-160 has a genome size of 1.22 Mbp and a genomic GC content of 42.4%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 38 tRNAs. It is auxotrophic for reduced sulfur sources, several amino acids (ornithine, histidine, betaine) and several vitamins (B1, B2, B5, B7, B12) and possesses rhodopsins. Members of the genus Nanopelagicus can be recognized by the presence of the diagnostic oligonucleotide sequence 5’-ACAAGAGGTTCGTCCGTCC-3’ in the 23S rRNA gene (positions 2669-2688, E. coli numbering). Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Nanopelagicus limnes MMS-21-122 (GCF_002287885.2) and Nanopelagicus abundans MMS-IIB-91 (GCF_002288305.1) with average amino acid identities of 84.5 and 80.7%, respectively, and average nucleotide identities of 78.6 and 76.2%, respectively.

Type genome is Nanopelagicus limnes MMS-21-122 (GCF_002287885.2), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.45±0.09 μm and diameters of 0.25±0.03 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Nanopelagicus limnes MMS-21-122 has a genome size of 1.24 Mbp and a genomic GC content of 41.5%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 38 tRNAs. It is auxotrophic for reduced sulfur sources, several amino acids (proline, ornithine, histidine, betaine) and several vitamins (B1, B2, B5, B7, B12) and possesses rhodopsins. Members of the genus Nanopelagicus can be recognized by the presence of the diagnostic oligonucleotide sequence 5’-ACAAGAGGTTCGTCCGTCC-3’ in the 23S rRNA gene (positions 2669-2688, E. coli numbering). Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Nanopelagicus hibericus MMS-21-160 (GCF_002288005.1) and Nanopelagicus abundans MMS-IIB-91 (GCF_002288305.1) with average amino acid identities of 84.5 and 81%, respectively, and average nucleotide identities of 78.6 and 77.1%, respectively.

Type genome is Planktophila dulcis MMS-IIA-65 (GCF_002288225.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.44±0.09 μm and diameters of 0.26±0.03 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Planktophila dulcis MMS-IIA-65 has a genome size of 1.35 Mbp and a genomic GC content of 48.0%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 40 tRNAs. It is auxotrophic for reduced sulfur sources, betaine, and several vitamins (B1, B5, B7, B12) and possesses rhodopsins. Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Planktophila rubra IMCC25003 (GCA_002284855) and Planktophila sulfonica MMS-IA-56 (GCF_002288065.1) with average amino acid identities of 89.6 and 89.4%, respectively, and average nucleotide identities of 82.1 and 81.9%, respectively.

Type genome is Planktophila lacusMMS-21-148 (GCF_002287925.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.41±0.10 μm and diameters of 0.30±0.06 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Planktophila lacusMMS-21-148 has a genome size of 1.46 Mbp and a genomic GC content of 47.5%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 41 tRNAs. It is auxotrophic for reduced sulfur sources, betaine, and several vitamins (B1, B5, B7, B12) and possesses rhodopsins. Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Planktophila aquatilis IMCC26103 (GCA_002284875) and Planktophila rubra IMCC25003 (GCA_002284855) with average amino acid identities of 84.1 and 74.4%, respectively, and average nucleotide identities of 77.9 and 72.6%, respectively.

We propose the genome of strain MMS-VB-114 (GCF_002288365.1) as type for Planktophila limnetica and as type of the genus Planktophila as the original strain was never genome-sequenced. According to the previous description of a mixed culture (MWH-EgelM2-3.acI)(Jezbera et al., 2009) that has a 100% identical 16S rRNA gene sequences with strain MMS-VB-114, we propose the name Planktophila limnetica for strain MMS-VB-114 and make an amended description based on a complete genome. Represented by strain MMS-VB-114, which was isolated from Lake Zurich, Switzerland. Curved rods with lengths of 0.38±0.10 μm and diameters of 0.25±0.06 μm (see Table S3, Figure S1 in Neuenschwander et al. 2018). The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with pyruvate, urea, vitamins, and amino acids. P. limnetica MMS-VB-114 has a genome size of 1.33 Mbp and a genomic GC content of 45.0 %. It is auxotrophic for reduced sulfur sources and vitamins (B1, B5, B7), and possesses rhodopsins (see Table S7 in Neuenschwander et al. 2018). A complete genome is available at Genbank (GCF_002288365.1). Phylogenetic analyses of 476 concatenated protein sequences, 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide identities and protein similarities indicated the affiliation of the taxon to the family Nanopelagicaceae and the order Nanopelagicales.

Type genome is Planktophila sulfonica MMS-IA-56 (GCF_002288065.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.50±0.14 μm and diameters of 0.28±0.06 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Planktophila sulfonica MMS-IA-56 has a genome size of 1.34 Mbp and a genomic GC content of 48.6%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 39 tRNAs. It is auxotrophic for reduced sulfur sources, betaine, and several vitamins (B1, B5, B7, B12) and possesses rhodopsins. Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Planktophila rubra IMCC25003 (GCA_002284855) and Planktophila dulcis MMS-IIA-65 (GCF_002288225.1) with average amino acid identities of 90.8 and 89.4%, respectively, and average nucleotide identities of 83.7 and 81.9%, respectively.

Type genome is Planktophila vernalis MMS-IIA-15 (GCF_002288185.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.43±0.12 μm and diameters of 0.26±0.05 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Planktophila vernalis MMS-IIA-15 has a genome size of 1.36 Mbp and a genomic GC content of 45.7%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 39 tRNAs. It is auxotrophic for reduced sulfur sources, serine, and several vitamins (B1, B5, B7, B12) and possesses rhodopsins. Planktophila vernalis can be recognized by the presence of the diagnostic oligonucleotide sequence 5’- AACTACTACCACACCGGTTCG-3’ in the 23S rRNA gene (positions 1420-1441, E. coli numbering). Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Planktophila sp. IMCC19121 (GCA_002284895) and Planktophila limnetica MMS-VB-114 (GCF_002288365.1) with average amino acid identities of 93.1 and 72.1%, respectively, and average nucleotide identities of 89.7 and 71.3%, respectively.

Type genome is Planktophila versatilis MMS-IA-79 (GCF_002288105.1), which was isolated via dilution-to-extinction cultivation from Lake Zurich, Switzerland. Curved rods with lengths of 0.45±0.10 μm and diameters of 0.27±0.04 μm. The initial pure culture was lost after a few propagations to fresh medium; no growing culture is available. The initial culture grew well in sterile lake water amended with minimal carbon medium, vitamins and amino acids. Planktophila versatilis MMS-IA-79 has a genome size of 1.33 Mbp and a genomic GC content of 48.2%. The genome is complete, consisting of a circular chromosome and contains 3 rRNA genes and 38 tRNAs. It is auxotrophic for reduced sulfur sources, betaine, and several vitamins (B1, B5, B7, B12) and possesses rhodopsins. Basis for the assignment is a phylogenomic tree of 476 concatenated protein sequences, phylogenetic analysis of 16S rRNA, 23S rRNA, and rhodopsin genes, as well as average nucleotide and amino acid identities presented in Neuenschwander et al. (2018, ISMEJ, doi: 10.1038/ismej.2017.156). The closest cultivate relatives are Planktophila dulcis MMS-IIA-65 (GCF_002288225.1) and Planktophila sulfonica MMS-IA-56 (GCF_002288065.1) with average amino acid identities of 82.4 and 82.0%, respectively, and average nucleotide identities of 76.8 and 76.2%, respectively.