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+Ccontents 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-ANTBAC T (=JCM 32977 T =LMG 31079 T =DSM 108553 T ) 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-WUEMPEL T (=LMG 31081 T =DSM 108554 T ).

The phylum Bacteroidetes contains Gram-negative bacteria, which occur in a broad variety of habitats all over the world and show different lifestyles and physiologies [1]. The Cytophaga-Flavobacteria cluster belongs to this phylum and is characterized by rod-shaped, non-spore-forming, frequently pigmented and gliding, chemoorganotrophic bacteria [2]. They are often capable of degrading biopolymers such as agar, cellulose, chitin, pectin and keratin [3]. Bacteria belonging to the Cytophaga-Flavobacteria cluster occur in high abundances in many freshwater and marine systems and play an important role in these ecosystems in respect to the uptake and degradation of dissolved organic material [2]. A large family within this cluster are the Cytophagaceae, which comprise 30 genera and about 150 species. Phylogenetic analyses based on 16S rRNA gene sequences suggested that this family encompass divergent clades which cannot be well distinguished from members of the families Cyclobacteriaceae and Flammeovirgaceae [4]. Genome-scale phylogenies inferred from whole proteomes revealed that the Cytophagaceae as a family and some of their genera are not monophyletic [1].
Within a cooperation project between schools and science (Sparkling Science program), which pursues the aim to isolate and taxonomically describe new bacterial species, 125 teenagers from high schools took samples from various freshwater habitats around the city of Salzburg (Austria), measured basic water chemistry parameters and inoculated agar plates with the collected water samples. By screening these plates, we found several strains belonging to an obviously undescribed genus affiliated with the family Cytophagaceae. From nine pure cultures, three were selected for further analysis. Some students joined the lab and helped with the phenotypic characterizations of the strains. Furthermore, some students created, with the help of a nomenclature advisor, the proposed genus and species names.
So, here we describe a new genus belonging to the Cytophagaceae which seems to be common in various aquatic freshwater habitats. As type species for this new genus we propose Aquirufa antheringensis gen. nov., sp. nov. with its type strain 30S-ANTBAC T . Furthermore, we propose to establish an additional species within this genus, Aquirufa nivalisilvae for strain 59G-WUEMPEL T .

HOME HABITATS AND ISOLATION
Strain 30S-ANTBAC T was isolated from Antheringer Bach, a small freshwater creek running through the town Anthering, Austria, the approximate geographic coordinates are 47.877 N 13.006 E. Water sampled in November 2017 had a pH of 6.9 and a conductivity of 435 µS cm À1 . Strain 103A-SOEBACH was isolated from Soellheimer Bach, a small freshwater creek located in Salzburg, Austria, the approximate geographic coordinates are 47.826 N 13.049 E. Water sampled in May 2018 had a pH of 6.9 and a conductivity of 515 µS cm À1 . Strain 59G-WUEMPEL T was isolated from a small intermittent freshwater pond located in a forest in Schneegattern (Lengau), Austria, the approximate geographic coordinates are 48.029 N 13.299 E. Water sampled in April 2018 had a pH of 7.5 and a conductivity of 163.7 µS cm À1 . All three strains were isolated by filtrating the samples and subsequent plating on agar plates. For strains 30S-ANTBAC T and 103A-SOEBACH filters with 0.45 µm pore size and for strain 59G-WUEMPEL T filters with 0.65 µm pore size were used. Nutrient broth-soyotone-yeast extract (NSY) agar plates [5] were utilized for 103A-SOEBACH, while Reasoner's 2A (R2A) agar plates [6] were used for strain 30S-ANTBAC T and tryptone-soyotone (DTS) plates [7] with very low nutrient concentrations were used for strain 59G-WUEMPEL T for the first cultivation steps, respectively. Nevertheless, all strains grew on NSY plates and in liquid NSY medium at pH 7.2 and were purified using these media.

PHENOTYPIC AND CHEMOTAXONOMIC CHARACTERIZATION
The temperature range for growth was tested on NSY agar plates exposed to increasing temperatures in 1 C steps starting at 5 C until no growth was observed. NaCl tolerance was tested using agar plates with various NaCl concentrations in 0.1 % (w/v) steps. For testing anaerobic growth, an anaerobic chamber and standard NSY agar plates as well as NSY plates supplemented with 2 g l -1 NaNO 3 were used. For determination of cell morphology and cell dimensions, well-growing liquid cultures were fixed with 2 % paraformaldehyde, stained with DAPI (4¢,6-diamidino-2-phenylindole) and investigated by using an epifluorescence microscope (UV filter). To test the ability of the strains to glide, soft agar plates (1 g l À1 yeast extract, 0.1 g l À1 K 2 HPO 4 , 2.0 g l À1 agar) were used [8]. One drop of a well-growing culture was placed in the centre of these test plates, as well as on standard NSY plates, respectively, incubated at room temperature and observed for several days. Assimilation of various substrates was tested using GEN III MicroPlates (Biolog), which detect utilization of substrates as electron donors by the subsequent reduction of a tetrazolium redox dye. Cells from well-growing liquid cultures were centrifuged and added to the inoculum medium so that the OD of the culture correspond to 0.07 at a wavelength of 590 nm. The absorption was measured with a Multiskan FC (Thermo Scientific) at a wavelength of 595 nm after 48 h incubation at 20 C. After subtracting the value of the negative control (without substrate), obtained values from 0.016 to 0.05 were regarded as weak utilization and for >0.05 as positive. The chemotaxonomic characterization of the strains included analyses of the composition of whole-cell fatty acids, polar lipids and respiratory quinones. They were carried out by the Identification Service, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. For all chemotaxonomic analyses cells were inoculated into liquid NSY medium and harvested after 3 days of growth by centrifugation. For the whole-cell fatty acid composition, an Agilent Technologies 6890 N instrument, the Microbial Identification System (MIDI) Sherlock version 6.1 and the TSBA 40 database were used as described by Sasser [9]. Polar lipids and respiratory quinones were extracted and analysed as described by Tindall [10,11] based on the method by Bligh and Dyer [12]. To investigate if the pigmentation of the cultures was caused by flexirubin, tests with 20 % KOH and 12 M HCl were performed [13].
Cells of all investigated strains were slim rods. They formed bright red, in older stages dark-red coloured, circular, and convex colonies with smooth surface on agar plates and showed a strong orange-red colouring in liquid media. Strain 59G-WUEMPEL T grew up to a temperature of 35 C, the other two strains stopped growing at lower temperatures ( Table 1). All strains only tolerated low salt concentrations, showed no anaerobic growth and were able to glide and showed spreading over the whole soft agar plates within periods of 7 days (Table 1). Despite various efforts according to Bernardet et al. [13] the flexirubin test was negative for all investigated strains, maybe caused by other overlaying pigments. All three investigated strains assimilated pectin and Tween 40 and showed weak assimilation of acetoacetic acid, glucuronamide, and D-fructose-6-PO 4 ( Table 1). Strain 59G-WUEMPEL T showed weak assimilation of additional substrates ( Table 1). All three investigated strains contained nearly the same fatty acids but in various amounts, C 15 : 0 was only found in strain 30S-ANTBAC T ( Table 2). The amounts of the major fatty acids differed remarkably between strain 30S-ANTBAC T and strain 103A-SOEBACH, which should be classified as the same species (see below). For example, iso-C 15 : 0 constituted 20.3 % and 39.5 % of fatty acids in strains 30S-ANTBAC T and 103A-SOEBACH, respectively (Table 2). These finding suggested, that the relative fatty acid amounts can vary a lot among strains belonging to the same species grown under comparable conditions. A study describing a new Polynucleobacter species (Betaproteobacteria) within the cryptic species complex PnecC based on six investigated isolates came to the same conclusion [14]. Regarding the polar lipids, all three strains presented here contained phosphatidylethanolamine and various unidentified polar lipids like aminolipids, phospholipids and aminophospolipids ( Table 2, Fig. S1, available in the online version of this article). While for strain 30S-ANTBAC T seven polar lipids could be separated, strain 59G-WUEMPEL T showed a more diverse pattern with three additional unidentified polar lipids. The main respiratory quinone for all three strains presented here was MK7. Additionally, strain 30S-ANTBAC T and strain 103A-SOEBACH contained traces of MK6 (Table 2).

GENOMIC CHARACTERIZATION
DNA extraction and genome sequencing were performed as described previously [15]. A shotgun library was paired-end sequenced on an Illumina MiSeq instrument (2Â300 bp). De novo assemblies were performed by using the software SPAdes version 3.13.0 [16], details are given in Table 3. The obtained genome sequences were annotated by the NCBI Prokaryotic Genome Annotation Pipeline and for further comparative analyses by the Integrated Microbial Genomes/ Expert Review (IMG/ER) annotation pipeline and incorporated in the IMG database [17]. The IMG Genome ID and GenBank accession numbers of all three investigated strains and strain HME7025 (Kim et al., 'Pseudarcicella sp.' HME7025 genome sequencing and assembly, unpublished data), which is closely related to the three investigated strains, are listed in Table 3.
The size of the genomes of strain 30S-ANTBAC T and strain 103A-SOEBACH was 2.5 Mbp, while strain 59G-WUEM-PEL T had a size of 3.1 Mbp (Table 3). All strains had relatively low G+C contents of less than 45 mol% (Table 3). Interestingly, members of the nearest relatives of the new genus had genome sizes twice as big (Table 4). Additionally, among 60 genomes of members of the family Cytophagaceae and related taxa, strain 30S-ANTBAC T and strain 103A-SOEBACH not only possessed the smallest genome size but also the highest coding density. While coding densities of the other 58 genomes ranged from 85.6 % to 93.5 % coding bases (average 89.5 %, median 89.4 %), these two genomes were characterized by a value of 94.8 %. On the other hand, the G+C content of their genomes of 43 mol% was close to the average of the other Cytophagaceae genomes (range 35-56 mol%, average 45 mol%), suggesting that the genomes of these two strains underwent evolutionary genome streamlining, which was decoupled from changes in G+C content.

Pigmentation Red Red Red
Flexirubin test À À À Assimilation of:    15,15¢-monooxygenase. But all four strains had genes putatively encoding enzymes for the complete biosynthesis of 7,8-dihydro-b-carotene. The latter one serves as substrate for the monooxygenase, which produces retinal, the cofactor for the light driven proton pump proteorhodopsin [18]. Furthermore, only strain 30S-ANTBAC T and strain 103A-SOEBACH possessed genes putatively encoding a catalaseperoxidase and a cytochrome c peroxidase, which could protect bacteria from oxidative stress.
All three investigated strains and strain HME7025 encoded genes annotated for the biosynthesis of various carotenoids ( Table 5). Concerning the ABC-type transport systems the four strains showed different patterns (Table 5). Regarding nitrogen metabolism the four strains showed some shared genes with an interesting distribution pattern among them. All strains except 103A-SOEBACH possessed a gene cluster, which encompassed genes putatively necessary for the uptake and assimilation of nitrate and nitrite ( Table 5). All strains except 30S-ANTBAC T possessed a gene cluster with genes putatively encoding enzymes responsible for the reduction of nitrous oxide to nitrogen, which is the last step of the dissimilatory denitrification (see below).

PHYLOGENY
Phylogenetic and phylogenomic reconstructions were performed by using almost full length sequences of the 16S rRNA gene and an aligned concatenated set of 119 single copy marker proteins [19], respectively.
For the phylogenetic tree in Fig. 1, 16S rRNA gene sequences from the type species of all genera currently classified as Cytophagaceae [1,20], of further species closely related to the investigated strains, of Prevotella melaninogenica serving as outgroup, and of the investigated strains were used. The sequence set was aligned by using the software MEGA X [21]. The trimmed alignment consisted of 1336 alignment positions. With the same software the data set was tested for the best fitting model and thus a maximum-likelihood phylogenetic tree was calculated using Kimura's two-parameter model [22] as follows: discrete gamma distribution (G=5), 500 replications, gaps were partial deleted, which resulted in 1306 used positions. In addition, neighbour-joining and maximum-parsimony trees were calculated by using the corresponding algorithms.  [23].
To improve the phylogenetic placement of the members of the new genus, genome data sets were used to calculate a RAxML tree [24]. Of the 120 protein-encoding genes recommended by Parks et al. [19], one gene (protein family TIGR00095) was not present in any of the available genomes of members of the Cytophagaceae. Sequences of the remaining 119 protein families were concatenated and aligned by using MAFFT [25]. This resulted in a sequence alignment of 50 907 amino acid positions. This alignment was filtered by GBLOCKS (version 0.91b) [26] for highly variable positions. The chosen settings resulted in reduction of the alignment to 37 796 positions (74 % of positions) in 556 selected blocks. A maximum-likelihood tree (Fig. 2) was reconstructed using RAxML 8.2.10 [24], as implemented on the CIPRES Science Gateway version 3.3 [27], under the Protein CAT model with auto selection of a protein substitution matrix (model settings PROTCATAUTO), and 100 bootstrapping iterations.
The phylogenetic reconstruction revised the placements of the members of the Cytophagaceae and confirmed the clade comprising members of the genus Flectobacillus, Arcicella, Pseudarcicella and the new taxon. The latter was now clearly separated from the other three genera. (Fig. 2).

ECOLOGY
The three strains presented in this study were isolated from either standing or running freshwater systems. All home habitats had in common that they were small ones. Thus, all habitats were in close contact to terrestrial systems. This corresponded with the fact that all strains are obviously able to degrade the polymer pectin, which is a component of the cell wall of terrestrial plants. The ability of all investigated strains to glide could also be a hint to their lifestyle in small and not permanent waters, which potentially allow them to move on moist surfaces. Strains 30S-ANTBAC T and 103A-SOEBACH could have, via proteorhodopsin, the opportunity to use sunlight as a supplementary energy source, which indicates that the strains live in at least temporarily light exposed habitats. All investigated members of the new genus-like taxon possessed a gene cluster putatively encoding for enzymes responsible for the reduction of nitrous oxide to nitrogen. This last step of the dissimilatory nitrate reduction is used by some bacteria to transfer protons across the membrane for energy conservation and was considered as the least oxygen tolerant step of nitrogen respiration [28]. However, a recent study [29] from a marine system revealed that N 2 O-consuming bacteria were also present and active in oxygenated surface water, this might be attributed to anoxic micro-environments created by particles. Even though the members of the new taxon grew aerobically, the potential ability to use N 2 O could also be an advantage at the water sediment interface of small or ephemeral water bodies, where anaerobic conditions may occur.
BLAST searches with the 16S rRNA gene sequences of 30S-ANTBAC T and 59G-WUEMPEL T revealed that the new genus-like taxon represents a common and widespread group of freshwater bacteria. Even when taking only the 100 % and 99 % hits into account, more than 100 sequences from uncultured and cultured bacteria originating from all over the world were reported (Fig.  S2). This included detections from various inland water systems like rivers, lakes, ponds, groundwater and lake sediments in the USA, Canada, the Arctic, China, Japan, Africa and Europe. For instance, sequences from environmental samples putatively belonging to the new genus-like taxon were found in high-altitude lakes of the eastern Tibetan plateau [30], the temperate Ipswich and Parker River in Massachusetts, USA [31], the humic lake Grosse Fuchskuhle, Germany [32], and in sediments of the freshwater Lake Kasumigaura, Japan [33]. Nevertheless, only a very few isolates were found in databases corresponding to the 16S rRNA genes from strains 59G-WUEMPEL T and 30S-ANTBAC T with identities of more  In the case of 59G-WUEMPEL T six strains sharing 16S rRNA gene similarities of >99.7 % were isolated from a mesotrophic lake in the Republic of Korea (strains HME7025, IMCC25912 and HMD1017), from the sediment of a freshwater lake in Japan [33], from an unknown source in China (strain D11), and from freshwater in Taiwan (strain CAR-16). In case of strain 30S-ANTBAC T , only two isolates sharing 16S rRNA gene similarities of >99.3 %, one from an artificial mesotrophic lake in the Republic of Korea (strain HME7208) and one from Lake Maarsseveen, The Netherlands (strain MI3-53), could be found. In the 16S rRNA gene tree, strains 30S-ANTBAC T , 103A-SOEBACH and 59G-WUEMPEL T formed their own clade, well separated from the closest related genera. With their nearest relative Pseudarcicella hirudinis they showed low 16S rRNA gene sequence similarities of less than 94.5 %. The separation of the clade formed by the new strains was confirmed and more pronounced by the phylogenomic tree (Fig. 2). Pairwise whole genome average nucleotide identity (gANI) values   calculated with the type strains of Pseudarcicella hirudinis and Arcicella aurantiaca were only slightly higher (71.8 %) than pairwise values calculated (i) for any of the three new strains and the P. hirudinis type strain (in all three cases about 69 %), and (ii) for any of the three new strains and the A. aurantiaca type strain (in all three cases 69-70 %). A comparison of the new taxon with the closest related genera Pseudarcicella, Arcicella and Flectobacillus is given in Table 4. The new taxon differed in pigmentation, in predominant fatty acid composition and in the combination of some other features from all other genera (Table 4). The patterns of the polar lipids of the three investigated strains (S1) and Pseudarcicella hirudinis [35] differed strongly. The most striking point is the genome size of the new taxon, which is approximately half of that of these nearest related genera. These points and the phylogenetic reconstructions ( Fig. 1, Fig. 2), as well as 16S rRNA gene similarities and gANI values suggested that these three strains represent a new genus, for which we propose the name Aquirufa gen. nov.

PROPOSAL OF THE NEW GENUS
To test which of the three investigated strains belong to the same species, pairwise gANI values were calculated (Table 3). While strains 30S-ANTBAC T and 103A-SOE-BACH had a gANI value of 96.5 % which is slightly higher than the proposed cut-off of 95-96 % separating two species [34,[36][37][38][39], indicated the remaining gANI values of rounded 71 % ( Table 3) that the three investigated strains represented two different species [34,[36][37][38][39]. These findings were confirmed by the phylogenomic reconstructions with multiple amino acid sequences (Fig. 2), which separated the proposed novel species on different branches with branch length, i.e. evolutionary distances, similar to those between other type strains belonging to the same genus within the family Cytophagaceae (Fig. 2). The pairwise gANI value between strains 59G-WUEMPEL T and HME7025 of 97.4 % suggested, that these strains need to be considered as members of the same species. At first glance, the results of the gene content concerning the nitrogen metabolism disagree with the classification of the investigated strains into two species, since strains 30S-ANTBAC T and 103A-SOEBACH classified as one species differed in possessing gene clusters putatively encoding for nitrate/ nitrite assimilation and nitrous oxide reduction, respectively (see above). According to the pangenome concept, these two gene clusters could be regarded as auxiliary genes representing parts of the flexible genome of the strains. Such genes could be exchanged by intra-or even interspecific horizontal gene transfer [15], which might explain, why the clusters are present or absent in strains belonging to the same species. In the case of the predicted assimilatory nitrate reductase, the gene sequence similarity between strains HME7025 and 59G-WUEMPEL T , probably belonging to a single species, was relatively high (97 %), while the latter and strain 30S-ANTBAC T showed 70 %. In contrast, for the predicted nitrous oxide reductase and flanking genes, the sequence similarity between strains HME7025 and 59G-WUEMPEL T was the same (97 %), while the latter and strain 103A-SOEBACH showed 88 % similarity of the genes. This value is much higher than the whole genome sequence similarity of 71 % between these two strains and could imply that for this gene cluster horizontal gene transfer across species boundaries might be possible.
Some features distinguished the three strains from each other. Only strain 59G-WUEMPEL T weakly assimilated sucrose, D-mannose, D-salicin and D-aspartic acid (Table 1). Furthermore, while strain 30S-ANTBAC T had only four unidentified polar lipids, strain 59G-WUEMPEL T had five. The two strains 30S-ANTBAC T and 103A-SOEBACH considered as belonging to the same species, shared highly similar G+C values but differed in this characteristic from the third strain. Cells form rods and grow chemoorganotrophically and aerobically. Colonies grown on NSY or R2A agar are bright red, in older stages dark red pigmented, circular and convex with smooth surface. Liquid cultures in NSY or R2A medium have an intense red-orange colouring. Cells are able to glide on soft agar. Major respiratory quinone is MK-7, predominant fatty acid is iso-C 15 : 0 , identified polar lipid is phosphatidylethanolamine. Based on phylogenetic reconstructions with 16S rRNA gene sequences and amino acid sequences obtained from 119 single copy genes, respectively, the genus belongs to the family Cytophagaceae. G+C content is in the range of 38-43 mol%. The type species of the new genus is Aquirufa antheringensis.
The type strain is 30S-ANTBAC T (=JCM 32977 T =LMG 31079 T =DSM 108553 T ), which was isolated from a small creek with medium conductivity and nearly neutral pH located in Anthering, Austria. The genome of the type strain is characterized by a size of 2.5 Mbp and a G+C content of 42.6 mol%.