NOTE Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703 DOI 10.1099/ijsem.0.001700

Description of forsetii sp. nov., a marine isolated from North Sea water, and emended description of Gramella gaetbulicola Cho et al. 2011

Irina Panschin,1† Mareike Becher,2† Susanne Verbarg,1 Cathrin Spröer,1 Manfred Rohde,3 Margarete Schüler,4 Rudolf I. Amann,2 Jens Harder,2 Brian J. Tindall1 and Richard L. Hahnke1,*

Abstract Strain KT0803T was isolated from coastal eutrophic surface waters of Helgoland Roads near the island of Helgoland, North Sea, Germany. The taxonomic position of the strain, previously known as ‘’ KT0803, was investigated by using a polyphasic approach. The strain was Gram-stain-negative, chemo-organotrophic, heterotrophic, strictly aerobic, oxidase- and catalase-positive, rod-shaped, motile by gliding and had orange–yellow carotenoid pigments, but was negative for flexirubin-type pigments. It grew optimally at 22–25 C, at pH 7.5 and at a salinity between 2–3 %. Strain KT0803T hydrolysed the polysaccharides laminarin, alginate, pachyman and starch. The respiratory quinone was MK-6. Polar lipids comprised phosphatidylethanolamine, six unidentified lipids and two unidentified aminolipids. The predominant fatty acids

were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1!7c and iso-C17 : 1!7c, with smaller amounts of iso-C15 : 0 2-OH, C15 : 0, anteiso-C15 : 0 and

C17 : 1!6c. The G+C content of the genomic DNA was 36.6 mol%. The 16S rRNA gene sequence identities were 98.6 % with Gramella echinicola DSM 19838T, 98.3 % with Gramella gaetbulicola DSM 23082T, 98.1 % with Gramella aestuariivivens BG- MY13T and Gramella aquimixticola HJM-19T, 98.0 % with Gramella lutea YJ019T, 97.9 % with Gramella. portivictoriae DSM 23547T and 96.9 % with Gramella marina KMM 6048T. The DNA–DNA relatedness values were <35 % between strain KT0803T and type strains with >98.2 % 16S rRNA gene sequence identity. Based on the chemotaxonomic, phenotypic and genomic characteristics, strain KT0803T has been assigned to the genus Gramella, as Gramella forsetii sp. nov. The type strain is KT0803T (=DSM 17595T=CGMCC 1.15422T). An emended description of Gramella gaetbulicolaCho et al. 2011 is also proposed.

The genus Gramella, with the type species Gramella echini- on this sampling station is given in Gerdts et al. [14]. The cola, was proposed by Nedashkovskaya et al. [1] and com- strain was cultivated on agar plates with artificial seawater prises species that were isolated either from coastal surface after Schut et al. [15], supplemented with a mixture of amino T  seawater (G. flava LMG 27360 [2] and G. planctonica C- acids, carbohydrates, alcohols and carboxylic acids at 25 C T CAMWZ-3 [3, 4]), from tidal flat sediment (G. portivictoriae [13]. In the aforementioned study, strain KT0803T was named T T UST040801-001 [5], G. gaetbulicola RA5-111 [6], G. aes- Cytophaga sp. KT0803 (NCBI taxon ID: 120668), but has T T tuarii BS12 [7], G. oceani CC-AMSZ-T [8], G. aestuarii- been studied for a long time as ‘Gramella forsetii’ (NCBI taxon vivens BG-MY13T [9] and G. aquimixticola MY13T [10], and T ID: 411154). Bauer et al. [16] showed, based on the genome of G. lutea YJ019 [11], or from sea urchins (G. echinicola KMM strain KT0803T, that this strain has a huge potential for sur- 6050T [1] and G. marina KMM 6048T [12]. face adhesion and hydrolyses glycosides and proteins using This study investigates the taxonomic position of strain glycoside hydrolases and peptidases, respectively. Further- KT0803T which was isolated by Eilers et al. [13] in August more, strain KT0803T lacks genes encoding for chitin degra- 1999 from coastal eutrophic surface waters (1 m depth) at the dation and for the assimilation of nitrate, nitrite and urea [16].   sampling station Helgoland Roads (54 09¢ N 7 52¢ E) near Using proteomics, three major polysaccharide utilization loci the island of Helgoland in the North Sea. More information [17] were detected in the genome of strain KT0803T enabling

Author affiliations: 1Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; 2Max Planck Institute for Marine Microbiology, Bremen, Germany; 3Helmholtz Centre for Infection Research, Braunschweig, Germany; 4Cell Biology and Electron Microscopy, University of Bayreuth, Bayreuth, Germany. *Correspondence: Richard L. Hahnke, [email protected] Keywords: North Sea; Flavobacteriaceae; polysaccharides; Helgoland. †These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KT0803T is AF235117, and for the complete genome CU207366 Three supplementary figures and two supplementary tables are available with the online Supplementary Material.

001700 ã 2017 IUMS

697 Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703 the strain to hydrolyse (i) a-1,4-glucans (e.g. glycogen, starch, Bacto Marine broth) supplemented with different azurin- amylose), (ii) laminarin-like polysaccharides and (iii) alginate- cross-linked (AZO-CL)-polysaccharides, casein and gelatin  like polysaccharides [18]. Furthermore, most of the TonB- at 25 C for up to 14 days. Each 200 µl well of a microtitre dependent transporters, carbohydrate active enzymes and plate was filled with a small portion of one of the AZO-CL- SusD-like proteins that were involved in the decomposition of polysaccharides, AZO-CL-casein (Megazym), charcoal-pec- these polysaccharides were not secreted into the medium but tin, -gelatin (chapter 15.3.32.3, method 3; [23] and 100 µl were membrane-bound [18]. Gliding motility and identifica- medium. Each well was inoculated with 100 µl of a starved tion of the associated genes of strain KT0803T were docu- culture or 100 µl medium as control. Susceptibility to antibi- mented by McBride and Zhu [19] using liquid-filled tunnel otics was tested using the diffusion method with discs con- taining ampicillin (10 µg ml–1), erythromycin (15 µg ml–1), slides and modified Cytophaga agar (DSMZ medium 172). –1 –1 T rifampicin (5–30 µg ml ), streptomycin (10–50 µg ml ), Here we describe strain KT0803 in relation to previously –1 –1 published species of the genus Gramella using a polyphasic tetracycline (30 µg ml ) and vancomycin (30 µg ml ). including phenotypic, chemotaxonomic and geno- Genomic DNA of strain KT0803T was isolated using the mic characteristics, 16S rRNA gene-based phylogeny, and Wizard Genomic DNA Purification Kit (Promega). Photo- DNA–DNA hybridization. metric DNA–DNA hybridization with strain KT0803T and Gramella gaetbulicola DSM 23082T were performed in Oxidase activity was tested using filter-paper discs (Sarto- duplicate as described by De Ley et al. [24] with the modifi- rius grade 388) impregnated with 1 % solution of N,N,N’, cations suggested by Huss et al. [25] using a Cary 100 Bio N’-tetramethyl-p-phenylenediamine (Sigma-Aldrich); the UV/visual spectrophotometer equipped with a Peltier- blue–purple colour after adding the cells indicated a posi- thermostat 6Â6 multicell changer and a temperature con- tive test. Catalase activity was tested by adding drops of 3 % troller with in situ temperature probe (Varian). Genomic H O to the cells; the observation of bubbles indicated a 2 2 DNA–DNA hybridizations (dDDH) were performed using positive test. The presence of flexirubin-type pigments was the Genome-to-Genome Distance Calculator (2.0), formula investigated according to the KOH test of Bernardet et al. 2 [26, 27]. The G+C content of the chromosomal DNA was [20]. Temperature optimum and pH optimum were determined in silico from the genome. Genes encoding car- determined on BD-Difco marine broth for temperatures   bohydrate active enzymes and peptidases were retrieved from 0–42 C in steps of 2.5 C and pH values from 3.7–9.0 from the genome using the CAZy [28, 29] and MEROPS in increments of 0.5. Salinity optimum was determined in [30] databases, respectively. medium with salinities from 0–7 % by mixing 4Â marine broth solution (without NaCl, MgSO4 and CaCl2) with dif- For cellular fatty acid analysis, fatty acid methyl esters were ferent volumes of a 10Â saline solution (per litre: 194.5 g extracted from cells of strain KT0803Tcultivated on marine  NaCl, 85.71 g MgCl2 6 H2O, 18 g CaCl2 2 H2O and MilliQ broth (DSMZ medium 514) at 25 C for 2 days. Cell samples  water). Gliding motility was investigated by the hanging- were harvested by centrifugation at 3000 r.p.m., 20 C for drop method after Bernardet et al. [20] and by plating a 20 min during exponential growth phase. Fatty acid methyl droplet of a fresh culture on marine broth and HaHa soft esters were prepared and analysed using the protocol of agar (0.3 % agar, w/v). Utilization of carbon compounds K€ampfer and Kroppenstedt [31], followed by gas chroma- and acid production were determined using API 20 NE and tography (Agilent 5890). The microbial identification stan- API 50 CHE strips (bioMerieux) and GEN II MicroPlates dard software package MIDI Sherlock (version 6.1) [32] was (Biolog) according to the manufacturers’ instructions, with used to automatically integrate the peaks, annotate the fatty the following modifications. For API tests, the API medium acids and determine the relative percentages using TSBA40 (AUX, API 50 CHB/E) was either (i) mixed 1 : 1 with 2Â and TSBA50 databases. Respiratory lipoquinones were artificial seawater, 0.5 ml 0.3 % agar (Bacto, BD) and 0.1 % extracted from freeze-dried cell material with methanol/ (w/v, final) yeast extract (Oxoid), or (ii) supplemented with hexane, separated into their functional classes by thin-layer 3 % (v/v, final) seawater (Biomaris). The GEN II Micro- chromatography (TLC) and analysed by reverse-phase Plates were prepared with cells washed twice with HaHa HPLC as described by Tindall [33, 34]. Polar lipids were medium and incubated in the same medium [21] without extracted from the methanolic/aqueous phase and cell mate- carbon sources at a cell density of 80 % transmittance. rial remaining after extraction of the respiratory lipoqui-  Plates and strips were incubated at 25 C for 1–3 days. Fur- nones [33, 34], separated by two-dimensional TLC and ther substrate tests were performed in HaHa medium with- identified as described by Tindall et al. [23]. out carbon sources and supplemented with 0.2 % (w/v, Strain KT0803T belongs to the genus Gramella and has 16S final) of selected carbohydrates incubated with strain  rRNA gene sequence identities of 98.6, 98.3, 98.1, 98.1, 98.0, KT0803T at 25 C, with growth monitored using a UV-VIS 97.9 and 96.9 % with G. echinicola KMM 6050T, G. gaetbuli- spectrophotometer (UV-1202; Shimadzu) at OD . 600 cola RA5-111T, G. aestuariivivens BG-MY13T, G. aquimixti- For polysaccharide and peptide hydrolysis, strains were cola HJM-19T, G. lutea YJ019T, G. portivictoriae DSM incubated in HaHa medium (12 mg l–1 carbon source mix, 23547T and G. marina KMM 6048T, respectively (Fig. 1). DSMZ medium 1564, Hahnke and Harder [22]) and marine The pairwise 16S rRNA gene sequence identities were below broth (6 g l–1 carbon source mix, DSMZ medium 514, BD- established thresholds of 98.2 % [35] and 98.7 % [36–38].

698 Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703

90/- Gramella marina KMM 6048T (AY753911) 67/76 Gramella portivictoriae UST040801-001T (DQ002871) Gramella aquimixticola HJM-19T (KR868710) Gramella echinicola KMM 6050T (AY608409) Gramella forsetii KT0803T (AF235117) Gramella aestuariivivens BG-MY13T (KM591916) Gramella gaetbulicola RA5-111T (GQ857650) 100/100 100/100 Gramella oceani CC-AMSZ-T T (KC169796) 85/95 Gramella planctonica CC-AMWZ-3 T (KC169794) Gramella flava JLT2011T (JX397931) Gramella aestuarii BS12T (JF751047)

Salegentibacter echinorum HD4T (JN040280) flavus Fg 69T (AY682200) Salegentibacter salinarum ISL-4T (EF612764) Salegentibacter agarivorans KMM 7019T (DQ191176) ACAM 48 T (M92279) Salegentibacter holothuriorum KMM 3524T (AB116148) Salegentibacter salarius ISL-6T (EF486353) Salegentibacter mishustinae KMM 6049T (AY576653) Salegentibacter chungangensis CAU 1289T (KC683477)

85/82 100/100 3 Zunongwangia 100/100 4 Mesonia

100/100 7 Gillisia

RAxML/MP 100/100 7 Psychroflexus 0.05

99/100 6 Salinimicrobium

Fig. 1. Phylogenetic tree of members of the genus Gramella and closely related genera of the family Flavobacteriaceae. The tree was inferred from 1289 aligned characters of the 16S rRNA gene sequence under the maximum likelihood (ML) and maximum parsimony (MP) criterion as previously described by Göker et al. [42]. The sequences of the LTP v. 121 database [43, 44] and from the GenBank database were aligned in ARB [45] using the SINA aligner [46] and were manually corrected. The branches are scaled in terms of expected number of substitutions per site. Numbers adjacent to the branches are support values from 1000 RAxML bootstrap repli- cates (left) and from 1000 maximum-parsimony bootstrap replicates (right) if >60 % [42]. Numbers in wedges represent the numbers of sequences. The tree was rooted using type strains of the genera Zunongwangia, Salegentibacter, Salinimicrobium, Psychroflexus, Gilli- sia and Mesonia.

T The DNA–DNA relatedness values between strain KT0803 (15.2 %) and anteiso-C15 : 0 (5.9 %), the hydroxy fatty acids T (=DSM 17595 ) and closely related type strains were 21.2 iso-C15 : 0 2-OH (6.8 %) and iso-C17 : 0 3-OH (10.2 %), the ±2.3 % (genomic) and 25.6 %/34.9 % (photometric), for G. straight-chain unsaturated fatty acids C16 : 1!7c (8.1 %) and echinicola DSM 19838T and G. gaetbulicola DSM 23082T, C17 : 1!6c (5.5 %), the branched-chain mono-unsaturated respectively. These DNA–DNA relatedness values were fatty acid iso-C17 : 1!7c (7.2 %), and the straight-chain satu- below the 70 % threshold [39] which is accepted for species rated fatty acid C15 : 0 (6.4 %). A comparison to other mem- delineation. bers of the genus Gramella is presented in Table 1. The The major (>5 % of the total) fatty acids of strain KT0803T polar lipid profile comprised phosphatidylethanolamine, were the branched-chain saturated fatty acids iso-C15 : 0 five unidentified lipids (L2–L6) and two unidentified

699 Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703

Table 1. Fatty acid profiles of species of the genus Gramella aminolipids (AL1, AL2), and one unidentified lipid Taxa: 1, strain KT0803T; 2, G. echinicola KMM 6050T; 3, G. gaetbulicola RA5- present in moderate amounts (L7) (Fig. S1, available in the T T T 111 ; 4, G. portivictoriae UST040801-001 ; 5, G. marina KMM 6048 ; 6, G. aes- online Supplementary Material). The only respiratory lipo- tuariivivens BG-MY13T; 7, G. aquimixticola HJM-19T. Data are from Nedash- kovskaya et al. [1, 12], Lau et al. [5], Cho et al. [6], Jeong et al. [7], Hameed quinone was MK-6, a common feature within the family et al. [8], Liu et al. [2], Shahina et al. [3], Park et al. [10] and Yoon et al. [11]. Flavobacteriaceae [40]. The G+C content of the genomic TR, <1 %; –, not detected. DNA was 36.6 mol%. Fatty acid 1§ 2 3 4 5 6 7 In HaHa medium strain KT0803T used as carbon and energy source L-arabinose, acetate, cellobiose, citrate, a- Saturated cyclodextrin, b-cyclodextrin, dextrin, D-fructose, D-galac- C15 : 0 6.4 7.1 –– 3.9 tose, N-acetyl-D-glucosamine, D-glucose, glycerol, lactate, C16 : 0 TR 5.8 1.1 1.4 lactose, maltose, D-mannose, melibiose, raffinose, sucrose Branched saturated – 3.7 and trehalose, but not D-mannitol, L-rhamnose, D-sorbitol iso-C – 1.4 – TR TR TR 14 : 0 and succinic acid. iso-C15 : 0 15.2 14.4 21.4 38.1 17.9 19.2 8.6

iso-C16 : 0 2.1 13.1 1.5 1.9 6.3 6.3 3.6 Polysaccharide, casein and gelatin hydrolysis by strain T T iso-C17 : 0 TR 1.5 1.7 – KT0803 and G. gaetbulicola DSM 23082 were investigated

anteiso-C15 : 0 5.9 7.6 4 6.2 8.6 9.6 19.6 in HaHa medium and BD-Bacto Marine broth (Fig. S2). T anteiso-C16 : 0 – 1.3 Strain KT0803 hydrolysed casein and gelatin, a common Unsaturated feature of the genus Gramella. The genomes of strain T T C15 : 1!6c 2.9 1.9 – 1.8 1.1 0.6 TR KT0803 , G. echinicola DSM 19838 and G. portivictoriae T C16 : 1!7c* 8.1 DSM 23547 comprised mainly metallo- and serine-pepti- – C16 : 1!6c and/or 11.4 16.7 8.8 11.6 9.4 5.8 dases (among 150 181 peptidases) with few differences in ! C16 : 1 7c and/or the set of peptidases (Table S1). All four Gramella strains † C15 : 0 2-OH hydrolysed pachyman and starch but did not hydrolyse chi- ! – C17 : 1 6c 5.5 3.6 1.7 3.6 3.4 TR tosan, pectin or cellulose (Avicel). Galactan was hydrolysed ! – C17 : 1 8c 1.9 1.8 by strain G. gaetbulicola DSM 23082T. The genome of strain ! – C18 : 1 5c TR 1.0 TR KT0803T encoded a greater number of carbohydrate active ! – C18 : 1 9c 1.7 enzymes (164) than the genomes of strains G. echinicola Branched mono- DSM 19838T (127) and G. portivictoriae DSM 23547T (119), unsaturated but did not differ significantly in the number of CAZy fami- iso-C15 : 1 G TR 1.2 – 2.1 1.0 1.7 lies (49 v. 48) (Table S1). Most of these CAZymes are found iso-C16 : 1 H 1.6 5.8 – TR 2.7 1.9 2.5 in all three genomes and thus might define the core set of iso-C !7c‡ 7.2 3.5 5.7 5.7 5.5 9.5 7.2 17 : 1 CAZymes among these strains, yet each genome contained anteiso-C !8c 3.2 2.0 –– 2.2 3.0 17 : 1 a unique set of CAZymes. Hydroxy

C15 : 0 2-OH 3.2 2.0 1.5 1.9 2.8 1.9 4.2 Gliding motility is a general feature of Gramella [1], how-

C17 : 0 2-OH 3.6 2.6 3.8 1.5 5.1 5.7 13.9 ever the mode of gliding differs between strains of the same T iso-C15 : 0 2-OH* 6.8 genus [41]. Strain KT0803 has all essential gliding motility T C16 : 0 3-OH TR 0.8 – genes, comparable to genes of G. echinicola DSM 19838 T iso-C15 : 0 3-OH 1.9 1.3 3.2 6.2 1.5 1.8 TR and G. portivictoriae DSM 23547 (Table S2). Gliding

iso-C16 : 0 3-OH 2.3 5.9 2.6 3.4 3.9 3.4 2.5 motility could not be observed by the hanging- T iso-C17 : 0 3-OH 10.2 6.7 22.5 11.2 12.4 15.9 6.2 drop method. Strain KT0803 showed an unusual mode of Unknown 13.566 3.7 gliding on marine broth soft agar. Small droplet-like colo- nies surrounded the initial colony, increasing in size with *Reported as summed feature 3 (C16 : 1!7c and/or iso-C15 : 0 2-OH), but contains two peaks, the retention times of which indicate that they can be increasing distance from the initial colony (Fig. S3). Further separated into their individual components C !7c and iso-C 2-OH. biochemical and physiological characteristics of strain 16 : 1 15 : 0 T †Typically reported as summed feature 3 containing fatty acids that KT0803 are presented in (Table 2) and in the species cannot be separated by the MIDI system as either (C16 : 1!6c and/or description. C16 : 1!7c or C16 : 1!7c and/or iso-C15 : 0 2-OH). That these peaks cannot be separated is factually incorrect and is a misinterpretation of the In conclusion, the phenotypic, chemotaxonomic and geno- T purpose of ‘summed features’ in the MIDI system. mic characteristics presented support that strain KT0803

‡Reported as iso-C17 : 1!9c, but may be misidentified and is iso-C17 : 1 represents a novel species within the genus Gramella, for !7c [47]. which the name Gramella forsetii sp. nov. is proposed. An §Data from this study. emended description of Gramella gaetbulicola is also pro- posed based on data obtained in this study.

700 Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703

Table 2. Phenotypic characteristics that significantly differentiate strain KT0803T from type strains of other species of the genus Gramella Strains: 1, KT0803T; 2, G. echinicola KMM 6050T; 3, G. gaetbulicola RA5-111T; 4, G. portivictoriae UST040801-001T; 5, G. marina KMM 6048T; 6, G. aes- tuariivivens BG-MY13T; 7, G. aquimixticola HJM-19T. All strains were positive for requirement of sodium ions; catalase activity; hydrolysis of aesculin and gelatin; enzyme activities of acid phosphatase, alkaline phosphatase, leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase(C8) and naphthol-AS-BI phosphohydrolase; utilization of D-glucose and sucrose; and gliding motility. All strains were negative for flexirubin- type pigments; hydrolysis of chitin; indole production; enzyme activity of a- fucosidase; acid production from adonitol, inositol, sorbitol, sorbose and xylose; and nitrate reduction. Data are from Nedashkovskaya et al. [1, 12], Lau et al. [5], Cho et al. [6], Panschin et al. [41]. +, Positive; À, negative; ND, not determined.

1* 2 3 4 5 6 7

NaCl concentration for growth (%, w/v) 0.13–6.0 1–15 3–10 1.0–6.0 0–15 0.5–12 0.5–8.0  Growth temperature ( C) 2–30 4–37 4–37 4–36 4–42 4–37 10–40 Hydrolysis of: Tween 80 – +† + +† + + – DNA + +† + –† – ND ND Galactomannan, arabinoxylan, xylan – +†–* +† ND ND ND HE-cellulose – +†–* +†– ND ND Enzyme activities a-Galactosidase – + + + + –– b-Galactosidase – + + – + –– a-Chymotrypsin – + + + + + – b-Glucuronidase ––––––– N-Acetyl-D-glucosaminidase + + + –– + – Trypsin – + + + + + + Lipase (C14) + + – + + – + Urease + ––––– ND Utilization of: Arabinose + + ND + + –– N-Acetyl-D-glucosamine – + –– + ND ND Inositol + –––– ND ND Mannitol + –– + – ND ND Sorbitol ––– + – ND ND Acetate + + + –––– Citrate + ––– + –– D-Lactose + – + –– ND ND g -Hydroxybutyric acid + –– + – ND ND L-Asparagine – + ––– ND ND Acid production from: N-Acetyl-D-glucosamine – + ––– ND ND L-Arabinose + ––– + ND ND Cellobiose + – + – + ND ND Citrate ––––– ND ND Glucose + + –– + ND ND Glycerol, mannitol + –––– ND ND Lactose + – + –– ND ND Maltose + + –– + ND ND Melibiose –––– + ND ND Raffinose + + –– + ND ND Sucrose – + –– + ND ND Susceptibility to tetracycline – + + + + + + DNA G+C content: HPLC (mol%) ND 39.6 39.1 39.9 40.0 38.9 48.0 Genome (mol%) 36.6* 36.9† ND 39.6† ND ND ND

*Data from this study. †Panschin et al. [41].

701 Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703

EMENDED DESCRIPTION OF GRAMELLA gluconate. Resistant to erythromycin, streptomycin, tetracy- GAETBULICOLA CHO ET AL. 2011 cline and vancomycin, but sensitive to ampicillin and rifampi- cin. Carbohydrate active enzymes and peptidases encoded in The description of Gramella gaetbulicola is as given by Cho the genome are listed in Table S1. The major (>5 % of the et al. [6], with the following amendments. Hydrolyses gela- total) fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1!7c, iso- tin, pachyman and galactan, but not arabinoxylan, galacto- C17 : 1!8c, iso-15 : 0 2-OH, C15 : 0, anteiso-C15 : 0 and C17 : 1!6c. mannan, xylan, hydroxyethyl-cellulose, Avicel-cellulose, The polar lipid profile comprises phosphatidylethanolamine, pectin, carrageenan, chitosan or agar. six unidentified lipids and two unidentified aminolipids. The only respiratory lipoquinone is MK-6. DESCRIPTION OF GRAMELLA FORSETII T T SP. NOV. The type strain is KT0803 (=DSM 17595 =CGMCC 1.15422T), isolated from surface seawater at the island Hel- Gramella forsetii (for.set¢i.i. N.L. gen. masc. n. forsetii of goland, German North Sea. The G+C content of the geno- Forseti, the god of justice and reconciliation in Scandinavian mic DNA of the type strain is 36.6 mol%. mythology who presided at the Thing and is associated with Helgoland, the German island from where the bacterium was isolated). Funding information I. P. and R. L. H. were supported by the Bundesministerium für Ernah-€ Cells are Gram-stain-negative, motile by gliding, non-spore- rung und Landwirtschaft No. 22016812 (PI: Brian J. Tindall). forming, 0.6–0.8Â1.5–3.0 µm rods, aerobic, oxidase- and cata- Conflicts of interest lase-positive. Nitrate and nitrite reduction is not observed. The authors declare that there are no conflicts of interest. Colonies are orange-yellow, convex, circular and translucent with entire edges and a shiny, smooth surface. Flexirubin-type References  1. Nedashkovskaya OI, Kim SB, Lysenko AM, Frolova GM, Mikhailov pigments are absent. Grows between 2–30 C, at pH 6.0–8.3 VV et al. Gramella echinicola gen. nov., sp. nov., a novel halophilic and with a salinity between 0.13–6.0 % (w/v) with optimum bacterium of the family Flavobacteriaceae isolated from the sea  growth between 22–25 C, at pH 7.5 and a salinity between 2– urchin Strongylocentrotus intermedius. Int J Syst Evol Microbiol 2005;55:391–394. 3 %. Hydrolyses pachyman, Tween 40, aesculin, gelatin, starch 2. Liu K, Li S, Jiao N, Tang K. Gramella flava sp. nov., a member of and DNA, but not galactan, agar, cellulose, pectin, carra- the family Flavobacteriaceae isolated from seawater. Int J Syst geenan, chitosan, arabinoxylan, galactomannan, xylan or Evol Microbiol 2014;64:165–168. Tween 80. Grows in HaHa medium with L-arabinose, acetate, 3. Shahina M, Hameed A, Lin SY, Lee RJ, Lee MR et al. Gramella cellobiose, citrate, a-cyclodextrin, b-cyclodextrin, dextrin, D- planctonica sp. nov., a zeaxanthin-producing bacterium isolated from surface seawater, and emended descriptions of Gramella fructose, D-galactose, N-acetyl-D-glucosamine, D-glucose, glyc- aestuarii and Gramella echinicola. Antonie van leeuwenhoek 2014; erol, lactate, lactose, maltose, D-mannose, melibiose, raffinose, 105:771–779. sucrose and trehalose, but not D-mannitol, L-rhamnose, D-sor- 4. Shahina M, Hameed A, Lin SY, Lee RJ, Lee MR et al. Gramella bitol and succinic acid. In the GN2 MicroPlate utilizes a- planctonica sp. nov. In List of New Names and New Combinations cyclodextrin, glycogen, Tween 40, Tween 80, N-acetyl-D- Previously Effectively, but not Validly, Published, Validation List no. 158. Int J Syst Evol Microbiol 2014;64:2184–2187. galactosamine, adonitol, cellobiose, D-fructose, gentiobiose, 5. Lau SC, Tsoi MM, Li X, Plakhotnikova I, Dobretsov S et al. Gra- myo-inositol, a-D-lactose, lactulose, D-mannose, melibiose, L- mella portivictoriae sp. nov., a novel member of the family Flavo- rhamnose, sucrose, trehalose, turanose, monomethyl-succi- bacteriaceae isolated from marine sediment. Int J Syst Evol nate, acetate, formate, D-gluconic acid, D-glucosaminic acid, Microbiol 2005;55:2497–2500. D-glucoronic acid, b-hydroxy-butyric acid, g-hydroxybutyric 6. Cho SH, Chae SH, Cho M, Kim TU, Choi S et al. Gramella gaetbuli- acid, succinic acid, succinamic acid, L-glutamic acid, glycyl L- cola sp. nov., a member of the family Flavobacteriaceae isolated from foreshore soil. Int J Syst Evol Microbiol 2011;61:2654–2658. glutamic acid, hydroxyl L-proline, g-aminobutyric acid, 7. Jeong SH, Jin HM, Jeon CO. Gramella aestuarii sp. nov., isolated DL a inosine, thymidine, - -glycerol phosphate and glucose 6- from a tidal flat, and emended description of Gramella echinicola. phosphate, but not dextrin, methyl pyruvate, p-hydroxyphe- Int J Syst Evol Microbiol 2013;63:2872–2878. nylacetic acid, a-ketovaleric acid, sebacic acid or L-asparagine. 8. Hameed A, Shahina M, Lin SY, Liu YC, Lai WA et al. Gramella oce- Possesses alkaline phosphatase, acid phosphatase, a-glucosi- ani sp. nov., a zeaxanthin-producing bacterium of the family Flavo- dase, cysteine arylamidase, esterase (C4), esterase lipase (C8), bacteriaceae isolated from marine sediment. Int J Syst Evol lipase (C14), leucine arylamidase, naphthol-AS-BI-phospohy- Microbiol 2014;64:2675–2681. b 9. Park JM, Park S, Won SM, Jung YT, Shin KS et al. Gramella aes- drolase, N-acetyl- -glucosaminidase and valine arylamidase tuariivivens sp. nov., isolated from a tidal flat. Int J Syst Evol activity, but not a-galactosidase, b-galactosidase, b-glucosi- Microbiol 2015;65:1262–1267. dase, b-glucuronidase, b-mannosidase, a-fucosidase, a-chy- 10. Park S, Kim S, Jung YT, Yoon JH. Gramella aquimixticola sp. nov., motrypsin or trypsin activity. Acid is produced from L- isolated from water of an estuary environment. Int J Syst Evol arabinose, cellobiose, D-fructose, galactose, D-glucose, lactose, Microbiol 2015;65:4244–4249. maltose, D-mannose, raffinose, sucrose, trehalose, D-turanose, 11. Yoon J, Jo Y, Kim GJ, Choi H. Gramella lutea sp. nov., a novel spe- cies of the family Flavobacteriaceae isolated from marine sedi- glycerol, D-mannitol and glycogen, but not from citrate, ment. Curr Microbiol 2015;71:252–258. fucose, gentiobiose, D-lyxose, D-mannose, melibiose, L-rham- 12. Nedashkovskaya OI, Kim SB, Bae KS. Gramella marina sp. nov., nose, D-ribose, sucrose, D-tagatose, xylose, N-acetyl-D-glucos- isolated from the sea urchin Strongylocentrotus intermedius. Int J amine, D-adonitol, arabitol, dulcitol, inositol, D-sorbitol or Syst Evol Microbiol 2010;60:2799–2802.

702 Panschin et al., Int J Syst Evol Microbiol 2017;67:697–703

13. Eilers H, Pernthaler J, Glöckner FO, Amann R. Culturability and in 31. Kampfer€ P, Kroppenstedt RM. Numerical analysis of fatty acid situ abundance of pelagic from the North Sea. Appl patterns of coryneform bacteria and related taxa. Can J Microbiol Environ Microbiol 2000;66:3044–3051. 1996;42:989–1005. 14. Gerdts G, Wichels A, Döpke H, Klings K-W, Gunkel W et al. 40- 32. Sasser M. Identification of bacteria by gas chromatography of cel- year long-term study of microbial parameters near Helgoland lular fatty acids. USFCC Newsl 1990;20:16. (German Bight, North Sea): historical view and future perspec- 33. Tindall BJ. Lipid composition of Halobacterium lacusprofundi. tives. Helgol Mar Res 2004;58:230–242. FEMS Microbiol Lett 1990;66:199–202. 15. Schut F, Prins RA, Gottschal JC. Oligotrophy and pelagic 34. Tindall BJ. A comparative study of the lipid composition of Halo- marine bacteria: facts and fiction. Aquatic Microbial Ecology 1997;12:177–202. bacterium saccharovorum from various sources. Syst Appl Microbiol 1990;13:128–130. 16. Bauer M, Kube M, Teeling H, Richter M, Lombardot T et al. Whole genome analysis of the marine ‘Gramella forsetii’ 35. Meier-Kolthoff JP, Göker M, Spröer C, Klenk HP. When should a reveals adaptations to degradation of polymeric organic matter. DDH experiment be mandatory in microbial taxonomy? Arch Environ Microbiol 2006;8:2201–2213. Microbiol 2013;195:413–418. 17. Sonnenburg ED, Zheng H, Joglekar P, Higginbottom SK, Firbank 36. Rosselló-Móra R, Amann R. Past and future species defini- SJ et al. Specificity of polysaccharide use in intestinal bacteroides tions for Bacteria and Archaea. Syst Appl Microbiol 2015;38: species determines diet-induced microbiota alterations. Cell 2010; 209–216. 141:1241–1252. 37. Stackebrandt E, Ebers J. Taxonomic parameters revisited: tar- 18. Kabisch A, Otto A, König S, Becher D, Albrecht D et al. Functional nished gold standards. Microbiol Today 2006;33:152–155. characterization of polysaccharide utilization loci in the marine 38. Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence Bacteroidetes ‘Gramella forsetii’ KT0803. ISME J 2014;8:1492– between average nucleotide identity and 16S rRNA gene sequence 1502. similarity for species demarcation of prokaryotes. Int J Syst Evol 19. McBride MJ, Zhu Y. Gliding motility and Por secretion system Microbiol 2014;64:346–351. genes are widespread among members of the phylum Bacteroi- 39. Rosselló-Mora R, Amann R. The species concept for prokaryotes. detes. J Bacteriol 2013;195:270–278. FEMS Microbiol Rev 2001;25:39–67. 20. Bernardet J-F, Segers P, Vancanneyt M, Berthe F, Kersters K 40. Bernardet JF. Family I. Flavobacteriaceae Reichenbach 1992b, et al. Cutting a gordian knot: emended classification and descrip- 327VP (Effective publication: Reichenbach 1989b, 2013.) emend. tion of the genus Flavobacterium, emended description of the Bernardet, Segers, Van- canneyt, Berthe, Kersters and Vandamme Family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (Basonym, Cytophaga aquatilis Strohl and Tait 1978). Int 1996, 145 emend. Bernardet, Nakagawa and Holmes 2002, 1057. J Syst Bacteriol 1996;46:128–148. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ et al. (editors). Bergey’s Manual of Systematic Bacteriology. The 21. Hahnke RL, Bennke CM, Fuchs BM, Mann AJ, Rhiel E et al. Dilu- Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, tion cultivation of marine heterotrophic bacteria abundant after a Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, spring phytoplankton bloom in the North Sea. Environ Microbiol Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes, – 2015;17:3515 3526. 2nd ed, vol. 4. New York, NY: Springer; 2011. pp. 106–314. 22. Hahnke RL, Harder J. Phylogenetic diversity of Flavobacteria iso- 41. Panschin I, Huang S, Meier-Kolthoff JP, Tindall BJ, Rohde M et al. lated from the North Sea on solid media. Syst Appl Microbiol 2013; Comparing polysaccharide decomposition between the type 36:497–504. strains Gramella echinicola KMM 6050T (DSM 19838T) and Gra- 23. Tindall BJ, Sikorski J, Smibert RA, Krieg NR. Chapter 15: pheno- mella portivictoriae UST040801-001T (DSM 23547T), and emended typic characterization and the principles of comparative systemat- description of Gramella echinicola Nedashkovskaya et al. 2005 ics. In: Reddy CA, Beveridge TJ, Breznak JA, Marzluf GA, Schmidt emend. Shahina et al. 2014 and Gramella portivictoriae Lau et al. TM and Snyder LR (editors). Methods for General and Molecular 2005. Stand Genomic Sci 2016;11:1–12. Microbiology, 3rd ed. Washington, DC: American Society of Microbi- ology; 2007. pp. 330–393. 42. Göker M, Cleland D, Saunders E, Lapidus A, Nolan M et al. Com- plete genome sequence of Isosphaera pallida type strain (IS1BT). 24. De Ley J, Cattoir H, Reynaerts A. The quantitative measurement Stand Genomic Sci 2011;4:63–71. of DNA hybridization from renaturation rates. Eur J Biochem 1970; 12:133–142. 43. Yarza P, Richter M, Peplies J, Euzeby J, Amann R et al. The All-Species Living Tree project: a 16S rRNA-based phylogenetic 25. Huss VA, Festl H, Schleifer KH. Studies on the spectrophotometric tree of all sequenced type strains. Syst Appl Microbiol 2008;31: determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 1983;4:184–192. 241–250.  26. Auch AF, Klenk HP, Göker M. Standard operating procedure for 44. Yarza P, Ludwig W, Euzeby J, Amann R, Schleifer KH et al. calculating genome-to-genome distances based on high-scoring Update of the All-Species living tree project based on 16s and segment pairs. Stand Genomic Sci 2010;2:142–148. 23s rRNA sequence analyses. Syst Appl Microbiol 2010;33:291– 299. 27. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and 45. Ludwig W, Strunk O, Westram R, Richter L, Meier H et al. ARB: a improved distance functions. BMC Bioinformatics 2013;14:60. software environment for sequence data. Nucleic Acids Res 2004; 32:1363–1371. 28. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V et al. The Carbohydrate-Active EnZymes database (CAZy): an 46. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W et al. SILVA: a expert resource for Glycogenomics. Nucleic Acids Res 2009;37: comprehensive online resource for quality checked and aligned D233–D238. ribosomal RNA sequence data compatible with ARB. Nucleic Acids 29. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Res 2007;35:7188–7196. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 47. Herzog P, Winkler I, Wolking D, Kampfer€ P, Lipski A. Chryseobac- 2013. Nucleic Acids Res 2014;42:D490–D495. terium ureilyticum sp. nov., Chryseobacterium gambrini sp. nov., 30. Rawlings ND, Waller M, Barrett AJ, Bateman A. MEROPS: the Chryseobacterium pallidum sp. nov. and Chryseobacterium molle database of proteolytic enzymes, their substrates and inhibitors. sp. nov., isolated from beer-bottling plants. Int J Syst Evol Nucleic Acids Res 2014;42:D503–D509. Microbiol 2008;58:26–33.

703