Four new members of the family Cytophagaceae: Chryseosolibacter histidini gen. nov., sp. nov., Chryseosolibacter indicus gen. nov., sp. nov., Dawidia cretensis, gen. nov., sp. nov., and Dawidia soli, gen. nov., sp. nov. isolated from diverse habitat

Four novel strains were isolated: PWU4T and PWU20T were both from soil in Germany, PWU5T was isolated from soil in India and PWU37T was obtained from sheep faeces collected on the Island of Crete. Cells of each were observed to be Gram-negative, strictly aerobic, rod shaped, and to grow optimally between 28 and 34 °C, between pH 7.0 and 8.0 and without the addition of NaCl. The strains were found to be catalase and oxidase-negative and able to grow on most mono- and disaccharides, a few polysaccharides and organic acids. Their predominant menaquinone was identified as MK-7. Their major fatty acids were identified as C16:1 ω7c (PWU4T and PWU20T) and C16:1 ω5c (PWU5T and PWU37T). The DNA G + C contents of strains PWU4T, PWU20T, PWU5T and PWU37T were determined to be 50.2 mol%, 51.6 mol %, 39.8 mol% and 53.8 mol%, respectively. The 16S rRNA gene sequence analysis revealed that the close relatives Ohtaekwangia koreensis 3B-2T and Ohtaekwangia kribbensis 10AOT share less than 93.8% sequence similarity. The strains were classified in two groups, where PWU4T and PWU20T share 93.0% sequence similarity, and PWU5T and PWU37T share 97.5% sequence similarity. However, the members of each group were concluded to represent different species based on the low average nucleotide identity (ANI) of their genomes, 69.7% and 83.8%, respectively. We propose that the four strains represent four novel species of two new genera in the family Cytophagaceae. The type species of the novel genus Chryseosolibacter is Chryseosolibacter histidini gen. nov., sp. nov. with the type strain PWU4T (= DSM 111594T = NCCB 100798T), whilst strain PWU20T (= DSM 111597T = NCCB 100800T) is the type strain of a second species, Chryseosolibacter indicus sp. nov. The type species of the novel genus Dawidia is Dawidia cretensis gen. nov., sp. nov. with the type strain PWU5T (= DSM 111596T = NCCB 100799T), whilst strain PWU37T (= DSM 111595T = NCCB 100801T) is the type stain of a second species, Dawidia soli sp. nov.


Introduction
The family Cytophagaceae was originally introduced in 1940 by Stanier (Stanier 1940) and it is the largest family within the order Cytophagales (Albers and Siebers 2014). Isolates of the family Cytophagaceae are Gram-stain negative chemoorganotrophic aerobic bacteria, but also a few anaerobes (Nakagawa 2011). Furthermore, they are widely distributed in nature such as soil (Hirsch et al. 1998;Kim et al. 2013;Zhang et al. 2009), freshwater (Baik et al. 2006;Maejima et al. 2020), airborne (Buczolits et al. 2002), dessert (Zhou et al. 2007) and a glacier field (Chaturvedi et al. 2005).
Since the 'golden age' of antibiotic discovery, members of the phylum Bacteriodetes including the classes Flavobacteriia and Cytophagia, have contributed as producers of antimicrobial bioactive compounds (Ikegami et al. 1990;Katayama et al. 1985;Okanya et al. 2011a, b;Park et al. 2008;Singh et al. 1982). During the screening of antimicrobial activity from members of the Reichenbach culture collection held at the Helmholtz Center for Infection Research (HZI) Germany, four novel bacterial strains (designated PWU4 T , PWU5 T , PWU20 T and PWU37 T ) were identified. The aim of the present study was to explore the taxonomic status of the four bacteria as novel species by using a polyphasic approach.

Material and methods
Isolation of bacterial strains and culture conditions Strains PWU4 T , PWU20 T and PWU37 T were isolated from soil samples collected in May 1990 at Braunschweig, Germany (52.22090N 10.50902E, PWU4 T ); in May 1989 at Lucknow, Uttar Pradesh, India (26.8684N 80.90979E, PWU20 T ); in September 1991 at Braunschweig (52.21501N 10.53329E, PWU37 T ); strain PWU5 T was isolated from sheep faeces with plant residues collected in July 1988 at Crete Island (35.2463N 25.09705E). The strains were isolated using a dilution method on agar plates following the protocol of Reichenbach (Reichenbach 1992), maintained in E medium and kept in this medium at − 80 °C for long-term preservation. Ohtaekwangia koreensis 3B-2 T KCTC23018 T and Ohtaekwangia kribbensis 10AO T KCTC23019 T , which were isolated by Yoon et al. (2011a), were used as references strains and were grown under the same culture conditions.

Morphological, physiological and chemotaxonomy analysis
Phenotypic characterisation was performed following the protocols described previously (Kim et al. 2013;Maejima et al. 2020;Yoon et al. 2011a). Morphological characteristics of the strains were observed using light microscopy (Zeiss Axio Scope A1. Microscope) with Axio-Vision Rel. 4.8 software. For physiological and chemotaxonomic tests, the four strains were grown without NaCl in E broth medium at their optimum pH and temperature of pH 7 and 30 °C for strain PWU4 T , pH 7 and 28 °C for strains PWU20 T and PWU5 T , pH 7.4-8.0 and 34 °C for strain PWU37 T . Growth at various temperatures, pH and NaCl concentrations was carried out aerobically on E agar medium. To determine the optimal temperature and pH for growth, duplicate plates were incubated at 4-44 °C and also at pH 5.0-9.5 as described previously (Mohr et al. 2018). Anaerobic growth was performed using E agar plates with Anaerocult P (Merck) in a candle jar (Jones 1981) for 3 weeks of incubation. Catalase and oxidase activities were performed according to Maejima et al. (2020) and the production of flexirubin-type pigments was tested according to Reichenbach (Reichenbach 1992). Carbon source utilisation assays were carried out in duplicate using E broth medium along with the Gen III MicroPlate system (Biolog) according to the manufacturer's protocol. Enzyme activities was assayed using the API ZYM (Humble et al. 1977) and API CAMPY (Huysmans et al. 1995) systems (bioMérieux), according to the protocols of the manufacturer. Antibiotic resistances was tested on E agar medium using the disc-diffusion plate method (Bauer et al. 1966).
For chemotaxonomic analysis, strains PWU4 T , PWU5 T , PWU20 T and PWU37 T were grown on M broth medium. Freeze-dried cells were prepared for detection of the major isoprenoid quinones, the polar lipids and the cellular fatty acids of the strains. In brief, 200 ml well-grown cultures were centrifuged at 9000 rpm for 10 min and the pellet was washed three times followed by centrifugation 9000 rpm for 10 min, and then freeze-drying for two days. The isoprenoid quinone was extracted according to Komagata and Suzuki (1988) and analysed further using HPLC (Agilent 1260 Series; Agilent technology USA). The polar lipids were determined by twodimensional TLC (Lechevalier et al. 1977;Minnikin et al. 1984). The Sherlock Microbial Identification System (MIDI) was used for identifying cellular fatty acids (Sasser 2009).

16S rRNA gene sequencing and phylogenetic analysis
The genomic DNA of each strain were extracted using an Invisorb Spin Plant mini kit (Stratec Molecular, Germany). After 5 days incubation at room temperature, cells were harvested from E broth medium and the pellet was further processed through DNA extraction following the manufacturer's protocol. The 16S rRNA genes was amplified using the bacterial universal primer set 27F and 1492R following the protocol of Mohr et al. (2018). After the PCR products were confirmed on 0.8% agarose gel, they were purified using the NucleoSpin Gel and PCR Clean up Kit (Macherey-Nagel, Düren, Germany). The 16S rRNA gene sequences of strain PWU4 T , PWU5 T , PWU20 T and PWU37 T were determined and compared to sequences from the GenBank7EMBL/DDBJ public database. The Genome to Genome Distance Calculator (GGDC 2.1) web server (Meier-Kolthoff et al. 2013), available at http:// ggdc. dsmz. de/ was used to infer the 16S rRNA phylogenetic relationships (Meier-Kolthoff et al. 2014). Briefly, after creating a multiple sequence alignment with MUSCLE (Edgar 2004), maximum likelihood and maximum parsimony trees were inferred with RAxML (Stamatakis 2014) and TNT (Goloboff et al. 2008), respectively. For maximum likelihood, rapid bootstrapping in conjunction with the autoMRE boot stopping criterion (Pattengale et al. 2010) and subsequent search for the best tree was used, while for maximum parsimony, 1000 bootstrapping replicates were used.

Genome sequence analysis
Draft genome sequences of each of the four strains were determined according to Mohr et al. (2018) and were submitted to the GenBank/EMBL/DDBJ public database. Automated genome annotation was performed using DFAST (Tanizawa et al. 2018). The average nucleotide identity (ANI) values between the genomes of the strains and their close relatives were calculated with the OrthoANIu algorithm using the EZ-Genome web services (Yoon et al. 2017). Digital DNA-DNA hybridization (dDDH) values were calculated using the GGDC 2.1 online service at http:// ggdc. dsmz. de/ distc alc2. php (Meier-Kolthoff et al. 2014). The phylogenomic analysis, matrix of AAI (Amino Acid Identity) and matrix of POCP (Percentage of Conserved Protein) were carried out using the EDGAR 3.0, a free bioinformatic platform available under https:// edgar3. compu tatio nal. bio. uni-giess en. de where the genome sequence data were uploaded and the output was visualized (Dieckmann et al. 2021).

Morphological, physiological and biochemical analyses
The cells of strains PWU4 T , PWU5 T , PWU20 T and PWU37 T were observed to be straight rods, 2.32-7.62 µm in length, to stain Gram-negative and to form yellow colonies on E medium (Okanya et al. 2011a, b).
The major respiratory quinone was identified as menaquinone MK-7 for all the strains, as is also found in the closely related genus Ohtaekwangia (Table 1) and almost all of the members of the phylum Bacteroidetes (Kim et al. 2013;Maejima et al. 2020;Yoon et al. 2011b) except the family Flavobacteriaceae, which have menaquinones of type 6 (MK6) (Albers and Siebers 2014). The major polar lipids of strains PWU4 T , PWU5 T , PWU20 T and PWU37 T were identified as phosphatidylethanolamine and an unidentified polar lipid. The fatty acid profiles of the four strains are shown in Table 2, along with those of the reference strains used in this study. Saturated and monounsaturated fatty acids with iso C 15:0 and C 16:1 ω7c were observed in all the strains including the reference strains. The major fatty acids of strains PWU4 T , PWU5 T , PWU20 T and PWU37 T were identified as C 16:1 ω7c (32.5%), C 16:1 ω5c (43.8%), iso C 15:0 (43.6%) and C 16:1 ω5c (38.5%), respectively. In contrast to strain PWU4 T where C 16:1 ω7c was found to be the major fatty acid, this fatty acid was much less abundant in strain PWU37 T (9.0%). Moreover, C 16:1 ω5c was found to be the major fatty acid in strains PWU5 T and PWU37 T but not in strains PWU4 T and PWU20 T . Albers and Siebers (2014) highlighted that branched, unsaturated or hydroxyl fatty acids represented the predominant cellular fatty acids in most members of the family Cytophagaceae.

Phylogenetic and genome analysis
The phylogenetic tree showed that strains PWU4 T , PWU5 T , PWU20 T and PWU37 T belong to the family Cytophagaceae and that they are closely related but distinct from members of the genus Ohtaekwangia (Figs. 1, 2). The four strains shared 91.63 to 97.82% 16S rRNA gene sequence similarity with each other (  .5-8.0 6.5-8.5 6.5-9.0 5.0-9.5 5.5-9.0 4.5-9.0 Optimal pH 7 7 7 7.4-8.0 6.5-7.5 6.5-7.5 NaCl tolerance (%NaCl, w/v)  Yarza et al. (2014) as being useful for delineating prokaryotic genera. The phylogenomic tree (Fig. 2), AAI matrix (Fig. 3) and POCP matrix (Fig. 4) confirmed the close relationships of the four strains in the two different genera. Strain PWU5 T and PWU37 T are grouped in the same genus and strain PWU4 T and PWU20 T together in a second genus. However, it was noted that between strains PWU4 T and PWU20 T share only 93.2% 16S rRNA similarity (Table 3) and differed in fatty acid profiles and GC content. At this point we cautiously place these two strains in the same genus based on the 16S rRNA, phylogenomic, AAI and POCP analysis but we noted that further analyses should be conducted.
Interestingly, in contrast to Qin et al. (2014), where the POCP values greater than 50% similarity provided evidence for placing species in the same genus, this threshold does not appropriate within the present study. Similarly other researchers revised the POCP genus threshold with higher values than 50% (Haba et al. 2021; Gupta 2019; Wirth and Whitman 2018). Approximately 65-70% and greater than 80% threshold may be more reasonable values for the genera containing strains PWU4 T and PWU20 T and strains PWU5 T and PWU37 T , respectively. However, Xu et al. (2020) highlighted that AAI analysis may be more acceptable to distinguish taxa at the genus level than ANI and POCP analysis.
The DNA G + C content of strains PWU4 T , PWU5 T , PWU20 T and PWU37 T were determined

Taxonomic conclusions
Morphological, biochemical, physiological and phylogenetic characteristics of strains PWU4 T , PWU5 T , PWU20 T and PWU37 T confirmed their position within the family Cytophagaceae. However, the obtained genetic data between strains PWU4 T , PWU5 T , PWU20 T , PWU37 T and related genera differentiated them from known genera of the family Cytophagaceae. Therefore, strains PWU4 T , PWU5 T , PWU20 T and PWU37 T should be classified in two new genera within the family Cytophagaceae. Chryseosolibacter (Chry.se.o.so.li.bac.ter. Gr. masc. adj. chryseos, golden; L. gen. n. soli, soil; L. neut. n. bacter, bacteria; N.L. neut. n. Chryseosolibacter, the color of colony bacteria is something like golden soil. Gram-stain negative, rod-shaped, asporogenous, non-motile, mesophilic, heterophilic and aerobic bacteria. Catalase and oxidase negative. Growth is observed on D-gluconic acid and methyl pyruvate. The major cellular fatty acids are iso-C 15:0 and C 16:1 ω7c. The major respiratory quinone is menaquinone-7 (MK-7). The major identified polar lipid is phosphatidylethanolamine. Member of the family Cytophagaceae. The type species is Chryseosolum histidini.
The type strain PWU4 T (= DSM 111594 T = NCCB 100798 T ) was isolated from a soil sample collected in May 1990 at Braunschweig, Germany (52.22090N 10.50902E). The 16S rRNA gene and wholegenome sequences of PWU4 T have been deposited in GenBank/EMBL/DDBJ under accession numbers MW182516 and JAHESF000000000, respectively.

Description of Chryseosolibacter indicus sp. nov.
Chryseosolum indicus (in'di.cus. L. neut. adj. indicus, India, the origin of the soil sample from which the type strain was isolated).

Description of Dawidia cretensis sp. nov.
Dawidia cretensis (cre.ten`sis. L. fem. adj. cretensis, Cretan, the source of the sample from the type strain was isolated).
The type strain PWU5 T (= DSM 111596 T = NCCB 100799 T ) was isolated from sheep faeces with plant residues collected in July 1988 on the Island of Crete (35.2463N 25.09705E). The 16S rRNA gene and whole-genome sequences of strain PWU5 T have been deposited in GenBank/EMBL/DDBJ under accession numbers MW182518 and JAHESE000000000, respectively.
Description of Dawidia soli sp. nov.
Dawidia soli (so.li. L. gen. n. soli, of soil, the source of the type strain).
The type strain PWU37 T (= DSM 111595 T = NCCB 100801 T ) was isolated from a soil sample collected in September 1991 at Braunschweig (52.21501N 10.53329E). The 16S rRNA gene and whole-genome sequences of strain PWU37 T have been deposited in GenBank/EMBL/