International Journal of Systematic and Evolutionary Microbiology

Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting endophytes isolated from the halophyte plant Arthrocnemum macrostachyum. --Manuscript Draft--

Manuscript Number: IJSEM-D-18-00195R1 Full Title: Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting endophytes isolated from the halophyte plant Arthrocnemum macrostachyum. Article Type: Taxonomic Description Section/Category: New taxa - Keywords: Halomonadaceae, Odiel marshes, halophilic, PGPB, heavy metals Corresponding Author: Maria del Carmen Montero-Calasanz School of Biology, Newcastle University Newcastly upon Tyne, UNITED KINGDOM First Author: Salvadora Navarro-Torre Order of Authors: Salvadora Navarro-Torre Lorena Carro Ignacio D. Rodríguez-Llorente Eloísa Pajuelo Miguel Ángel Caviedes José Mariano Igual Susana Redondo-Gómez Maria Camacho Hans-Peter Klenk Maria del Carmen Montero-Calasanz Manuscript Region of Origin: SPAIN Abstract: Two endophytic (EAod3T and EAod7T) were isolated from the aerial part of plants of Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). Phylogenetic analysis based on 16S rRNA gene sequences indicated their affiliation to the genus Kushneria. 16S rRNA gene sequences of strains EAod3T and EAod7T showed the highest similarity with K. marisflavi DSM 15357T (99.0% and 97.6%, respectively). Digital DNA-DNA hybridization studies between the draft genomes of strain EAod3T and K. marisflavi DSM 15357T corresponded to 28.5% confirming the novel lineage of strain EAod3T in the genus Kushneria. Both strains were Gram staining-negative, aerobic and motile rods able to grow at 4-37ºC, at pH 5.0-8.0 and tolerate 0.5-25% NaCl (w/v). They presented ubiquinone Q9 and C16:0, C16:1ω7c/ C16:1ω6c and C18:1ω7c as the major fatty acids. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on the phenotypic and phylogenetic results, strains EAod3T (=CECT 9073T=LMG 29856T) and EAod7T (=CECT 9075T=LMG 29858T) are proposed as new representatives into the genus Kushneria, and the proposed names are K. phyllosphaerae sp. nov. and K. endophytica sp. nov., respectively. The whole genome sequence of strain EAod3T has a total length of 3.8 Mbp and a G+C content of 59.3 %.

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1 Kushneria phyllosphaerae sp. nov. and Kushneria endophytica

2 sp. nov., plant growth promoting endophytes isolated from

3 the halophyte plant Arthrocnemum macrostachyum.

4

5 Salvadora Navarro-Torre1, Lorena Carro2, Ignacio D. Rodríguez-Llorente1,

6 Eloísa Pajuelo1, Miguel Ángel Caviedes1, José Mariano Igual3, Susana

7 Redondo-Gómez4, Maria Camacho5, Hans-Peter Klenk2 & Maria del

8 Carmen Montero-Calasanz2*

9 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de 10 Sevilla, Calle Profesor García González, 2, 41012 Sevilla, Spain.

11 2School of Natural and Environmental Sciences (SNES), Newcastle University, 12 Newcastle upon Tyne, NE1 7RU, UK.

13 3Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de 14 Investigaciones Científicas (IRNASA-CSIC), c/Cordel de Merinas 40-52, 37008 15 Salamanca, Spain.

16 4Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de 17 Sevilla, 1095, 41012 Sevilla, Spain.

18 5IFAPA-Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres- 19 Tomejil, Ctra. Sevilla-Cazalla de la Sierra, Km 12.2, 41200 Alcalá del Río, Sevilla, 20 Spain.

21 *Corresponding author: María del Carmen Montero-Calasanz, Tel.: +44 22 (0)191.208.4943 e-mail: [email protected]

23 Running title: Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov.

24

25 Subject category: New taxa (Proteobacteria)

26 27 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of 28 strains EAod3T and EAod7T are KU320856 and KU320860, respectively. The 29 GenBank/EMBL/DDBJ accession number for the draft genome sequence of strain 30 EAod3T is ONZI01.

31

32 Abstract

33 Two endophytic bacteria (EAod3T and EAod7T) were isolated from the aerial part of 34 plants of Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). 35 Phylogenetic analysis based on 16S rRNA gene sequences indicated their affiliation to 36 the genus Kushneria. 16S rRNA gene sequences of strains EAod3T and EAod7T showed 37 the highest similarity with K. marisflavi DSM 15357T (99.0% and 97.6%, respectively). 38 Digital DNA-DNA hybridization studies between the draft genomes of strain EAod3T 39 and K. marisflavi DSM 15357T corresponded to 28.5% confirming the novel lineage of 40 strain EAod3T in the genus Kushneria. Both strains were Gram staining-negative, 41 aerobic and motile rods able to grow at 4-37ºC, at pH 5.0-8.0 and tolerate 0.5-25%

42 NaCl (w/v). They presented ubiquinone Q9 and C16:0, C16:1ω7c/ C16:1ω6c and C18:1ω7c 43 as the major fatty acids. The predominant polar lipids were diphosphatidylglycerol, 44 phosphatidylglycerol and phosphatidylethanolamine. Based on the phenotypic and 45 phylogenetic results, strains EAod3T (=CECT 9073T=LMG 29856T) and EAod7T 46 (=CECT 9075T=LMG 29858T) are proposed as new representatives into the genus 47 Kushneria, and the proposed names are K. phyllosphaerae sp. nov. and K. endophytica 48 sp. nov., respectively. The whole genome sequence of strain EAod3T has a total length 49 of 3.8 Mbp and a G+C content of 59.3 %. 50

51 Keywords: Halomonadaceae, Odiel marshes, halophilic, PGPB, heavy metals

52

53

54

55 56 The genus Kusnheria is a monophyletic group belonging to the family Halomonadaceae 57 [1] in the class . It was first suggested by Sánchez-Porro et al. [2] 58 to accommodate the species Kusnheria avicenniae [2, 3], Kushneria indalinina [2, 4] 59 and Kushneria marisflavi [2, 5] previously classified into the genus . At the 60 time of writing it comprises seven validly named species [6]: Kushneria aurantia (type 61 species) [2], K. avicenniae [2, 3], K. indalinina [2, 5], Kushneria konosiri [7], K. 62 marisflavi [2, 5, 8], Kushneria pakistanensis [9] and Kushneria sinocarnis [10]. 63 Representatives of the genus Kushneria are Gram-staining-negative, motile, non- 64 sporulating, halophilic and aerobic rods [2], presenting the ubiquinone Q9 as the major

65 respiratory quinone and C16:0, C18:1ω7c, C19:0cyclo ω8c and C12:0 3-OH as the 66 predominant fatty acids [2].

67 Recently, endophytic strains EAod3T and EAod7T were isolated from the aerial part of 68 the halophyte plant Arthrocnemum macrostachyum in the Odiel marshes in Huelva 69 (Spain) (37º 13’N - 6º 57’O) (see Navarro-Torre et al. [11] for details). According to 70 Navarro-Torre et al. [11], both isolates were able to grow in presence of high 71 concentrations of heavy metals and salt, tolerate up to 20 mM Pb, 23 mM Ni and 3 M 72 NaCl, and hydrolyse starch (amylase activity) and DNA (DNAse activity). Additionally 73 the presence of the enzyme caseinase was also observed for strain EAod3T. They 74 besides showed in vitro plant growth promoting (PGP) properties such as siderophores 75 production, indolacetic acid (IAA) production, nitrogen fixation and phosphate 76 solubilisation, in absence and presence of moderate amounts of heavy metals [11]. 77 Strain EAod3T also demonstrated a promising biotechnological potential as plant 78 growth and bioremediation promoter when inoculated as part of a bacterial consortium 79 on plants of A. macrostachyum under greenhouse conditions [12]. 80 In the present study, a polyphasic characterization, following the minimal standards for 81 the family Halomonadaceae [13], was performed to determine the taxonomic status of 82 both strains. Based on phylogenetic and phenotypic data, strains EAod3T and EAod7T 83 are proposed as new species within the genus Kushneria, for which the names 84 Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov. are proposed. 85 86 Briefly, endophytes from the aerial part of A. macrostachyum were isolated on Tryptic 87 Soy Agar (TSA) supplemented with 0.3M NaCl and grown at 28ºC for 72 h and then, 88 different strains were sorted by colony morphology and colour (see Navarrro-Torre et 89 al. [11] for details]. Pure cultures were preserved at -80ºC in 15% glycerol. 90 DNA extraction, 16S rRNA gene sequences amplification and sequencing were carried 91 out as outlined by Navarro-Torre et al. [11]. 16S rRNA gene sequences were deposited 92 in GenBank/EMBL/DDBJ data library and aligned with corresponding sequences of 93 closely related type strains retrieved by Ez-Taxon-e service 94 (http://www.ezbiocloud.net/eztaxon) [14]. Genome from strain EAod3T was sequenced 95 and assembled by MicrobesNG company (Birmingham, United Kingdom) using 96 Illumina technology and a standard analysis pipeline. The closest available reference 97 genome was identified using Kraken [15], the quality of data was assessed mapping the 98 reads to this using BWA mem [16], de novo assembly of the reads was done using 99 SPAdes [17] maping the reads back to the resultant contigs, and again using BWA mem 100 to get more quality metrics. The whole draft genome was deposited in 101 GenBank/EMBL/DDBJ. RAST server v2.0 [18], QUAST v.4.6.3 software [19], 102 PROKKA [20], SignalP 4.1 server [21], TMHMM server v.2.0 [22], and CRISPRFinder 103 [23] were used for genome annotation and getting basic genome statistics. 104 Digital DNA-DNA hybridization (dDDH) tests between strain EAod3T and K. 105 marisflavi DSM 15357T [24] was performed using GGCD web server [25] available at 106 http://ggdc.dsmz.de/ . A phylogenetic tree was inferred as previously described [26] 107 using the aforementioned server [25, 27]. Pairwise sequence similarities were calculated 108 using the method of Meier-Kolthoff et al. [28]. 109 Temperature (4, 15, 20, 25, 28, 30, 32, 37 and 45ºC) and pH (4.5, 5.0, 6.0, 7.0, 8.0, 8.5 110 and 9.0) ranges were studied on TSA plates supplemented with 0.3M NaCl for 6 days 111 and at 28ºC. The pH values were adjusted with citrate-phosphate buffers (0.1M citric 112 acid and 0.2M dibasic sodium phosphate) and Tris-HCl buffer (0.1M Tris 113 (hydroxymethyl) aminomethane and 0.1M HCl). The NaCl tolerance was performed 114 using mTGE agar plates [9] at several concentrations of NaCl (0, 0.5, 2.5, 5, 7.5, 10, 115 12.5, 17.5, 20, 25 and 30%; w/v) for 6 days at 28ºC. The growth under anaerobic 116 conditions was carried out using TSA semisolid tubes containing 2.5% NaCl (w/v) and 117 2% agar (w/v) and sealed with paraffin at 30ºC for 10 days [10]. In addition, growth 118 characteristics were tested on marine agar 2216 (MA) and the selective media 119 MacConkey and Cetrimide agar supplemented with 2.5% NaCl (w/v) for 48 h at 28ºC. 120 The colony morphology was observed under a stereoscopic microscope (Olympus 121 SZ61) on TSA plates supplemented with 2.5% NaCl (w/v) at 30ºC for 48 h. The colony 122 colour was determined by comparing with RAL D2 Design colour chart. The cell 123 morphology was observed after Gram staining [29] using an Olympus CX41 124 microscope (objective 100X). The motility was tested by observing a drop of bacterial 125 suspension growing in TSB 0.3M NaCl at 28ºC for 30 min under optical microscopy 126 (objective 100X) [11]. Biochemical and enzymatic characteristics were tested using API 127 20NE, API 20Strep and API ZYM galleries (bioMérieux, France) according to the 128 manufacturing’ instructions. Oxidase activity was performed by adding 1% tetramethyl- 129 p-phenylenediamine reagent (Becton, Dickinson and Company, Mexico) to a loop of 130 bacterial biomass. The test was positive when the biomass became blue after 10-15

131 seconds. For the catalase activity, 3% H2O2 was added to a colony. The production of 132 bubbles indicated that the test was positive. Oxidation of carbon compounds and 133 sensitivity to some antimicrobial agents were determined using GEN III MicroPlates 134 (BIOLOG) incubated in an Omnilog device (BIOLOG) for 3 days at 30 ºC. Viscous 135 inoculating fluid C supplemented with 0.3M NaCl was used to inoculate the strains at 136 90–95 % transmittance. Parallel assays were performed with the reference strains K. 137 indalinina CECT 5902T, K. marisflavi DSM 15357T and K. pakistanensis KCTC 138 42082T. The results were analysed with the opm package for R [30, 31] v.0.9.23. 139 Chemotaxonomic analyses were performed using freeze-dried biomass except cellular 140 fatty acids analysis. For fatty acids extraction, strains EAod3T and EAod7T along the 141 previously mentioned reference strains were grown on TSA plates with 2.5% NaCl 142 (w/v) for 48 h at 30ºC and then, 40 mg of bacterial biomass was harvested to proceed 143 with the extraction as outlined by Sasser [32]. Results were analysed using the 144 Microbial Identification System (MIDI) Sherlock Version 6.1 (TSBA40 database). 145 Respiratory quinones were extracted using methanol:hexane (2 : 1, vol/vol), separated by 146 TLC and determined by HPLC [33, 34]. Polar lipids were extracted and separated by 147 two-dimensional TLC following the protocol established by Minnikin et al. [35] and 148 modified by Kroppenstedt and Goodfellow [36]. Polar lipids were identified as outlined 149 by Tindall [33, 34]. 150 151 16S rRNA gene sequences of strains EAod3T and EAod7T (1467 and 1418 bp, 152 respectively) showed the highest similarity with K. marisflavi DSM 15357T (99.0%), K. 153 indalinina CECT 5902T (98.6%), and K. pakistanensis KCTC 42082T (98.4%) and K. 154 marisflavi DSM 15357T (97.6%), K. indalinina CECT 5902T (97.3%) and K. 155 pakistanensis KCTC 42082T (96.9%), respectively. 16S rRNA gene sequence similarity 156 between strains EAod3T and EAod7T was 98.4%. The inferred phylogenetic tree 157 unambiguously showed strains EAod3T and EAod7T within the genus Kushneria (Fig. 158 2). Additionally Maximum Likelihood and Maximum Parsimony estimations supported 159 the separation of the two strains into two lineages with maximum support. Based on the 160 observations reported by Meier-Kolthoff et al. [28] who statistically established a 16S 161 rRNA threshold at 98.7 % for Proteobacteria with a maximum probability of error of 162 1.00 % to get DNA–DNA hybridization values above the 70 % threshold recommended 163 by Wayne et al. [37], to confirm the species status of novel strains, dDNA-DNA 164 hybridization studies were only carried out between the draft genomes of strain EAod3T 165 and K. marisflavi DSM 15357T and corresponded to 28.5%. Its value confirmed the 166 novel lineage of strain EAod3T in the genus Kushneria. The whole genome sequence of 167 strain EAod3T has a total length of 3,764,595 bp and a G+C content of 59.3 % (see 168 Supplementary Table S1 for additional details). 169 170 Cells of strains EAod3T and EAod7T were Gram-staining-negative, motile, non-spore 171 forming and aerobic rods of 0.1x0.2-0.4 µm (in growth phase) (Supplementary Fig. S1). 172 These characteristics are in agreement with the original description of genus Kushneria 173 [2]. The bacterial colonies of both strains were light salmon in colour (RAL 060 80 40) 174 smooth in surface, opaque, viscous, and convex with an undulate margin. Optimal 175 growth conditions for the two strains were on TSA plates with 2.5% NaCl (w/v) and pH 176 of 6.0-8.0 at 30ºC for 48 h. Nevertheless, the two strains were able to grow at 4-37ºC 177 (growth at 4ºC was poor), at pH 5.0-8.0 and tolerate 0.5-25% NaCl (w/v). According to 178 these results, strains EAod3T and EAod7T could be considered moderately halophilic 179 microorganisms [38]. The two strains grew well on MA plates and MacConkey agar 180 accordingly to results described for other species in the genus [9, 10, 14] but not on 181 cetrimide agar. When comparing with the reference strains, both strains showed 182 differential phenotypic features as shows in Table 1 and Supplementary Fig.S2. 183 184 As previously reported for the genus Kushneria [2], predominant fatty acids of strains T T 185 EAod3 and EAod7 were C18:1ω7c (44.8% and 44.6%, respectively), C16:0 (26.4% and

186 27.0%, respectively), and C16:1ω7c/ C16:1ω6c (13.7% and 13.0%, respectively) (Table 2), 187 the major respiratory quinone was ubiquinone 9 (Q9; 84.4% and 85%, respectively) and 188 polar lipids pattern of both strains comprised phosphatidylglycerol (PG), 189 diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE) (Fig. 1). In minor 190 extension, both strains presented an unidentified aminoglycolipid (AGL) and several 191 unidentified phospholipids. An unidentified aminoglycolipid presenting the same Rf 192 value was previously described in other species in the genus [2]. 193 194 On basis of the phenotypic and genotypic similarities and differences obtained in this 195 work, the strains EAod3T and EAod7T are proposed as novel species in the genus 196 Kushneria.

197

198 DESCRIPTION OF KUSHNERIA PHYLLOSPHAERAE SP. NOV.

199 Kushneria phyllosphaerae (phyl.lo.sphae′rae. Gr. neut. n. phyllon leaf; L. fem. n. 200 sphaera ball, sphere; N.L. gen. n. phyllosphaerae of the phyllosphere, as the bacterium 201 was isolated from the phyllosphere of a plant). 202 Cells are Gram-staining-negative, motile, aerobic, 0.1x0.2-0.3 µm in size, non-spore 203 forming rods frequently appearing as single short rods. Bacterial colonies are light 204 salmon in colour (RAL 060 80 40), smooth, opaque, convex, with an undulate margin 205 and 3 mm in size when growing on TSA supplemented with 2.5% NaCl (w/v), pH 7.0 at 206 30ºC for 2 days. Growth occurs at temperatures of 4-37ºC (optimum 30ºC), although the 207 growth at 4ºC is very weak. The pH range is 5.0-8.0 (optimum pH 7.0). Can tolerate 208 until 25% NaCl (w/v) and needs, at least, 0.5% NaCl (w/v), so it is a moderately 209 halophilic microorganism. Does not growth on cetrimide agar. Grows on MacConkey 210 agar and MA. Catalase positive and oxidase negative. Starch, casein and DNA are 211 hydrolysed while Tween 80, chitin, cellulose and pectin are not. Strong enzymatic 212 activity for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine 213 arylamidase, acid phosphatise, α-glucosidase and β-glucosidase; weak enzymatic 214 activity for lipase (C14), α-chymotrypsin, naphthol-AS-BI-phosphohydrolase and β- 215 galactosidase (para-nitroPhenyl β-D-galactopyranosidase); and negative for valine 216 arylamidase, cysteine arylamidase, trypsine, α-galactosidase, β-glucoronidase, N-acetyl- 217 β-glucosaminadase, α-mannosidase, α-fucosidase, arginine dihydrolase, urease, 218 pyrrolidonyl arylamidase (PYR) and β-glucuronidase. Positive for the hydrolysis of 219 esculin and production of acetone (Voges Proskauer positive); negative for the 220 hydrolysis of gelatin and indole production. Can reduce nitrates to nitrites, ferment D- 221 glucose, assimilate potassium gluconate and malic acid, but cannot assimilate D- 222 glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, 223 capric acid, adipic acid, trisodium citrate and phenylacetic acid. Acid is produced from 224 D-trehalose, but not from D-ribose, L-arabinose, D-mannitol, D-sorbitol, D-lactose, 225 inulin, D-raffinose, starch and glycogen. The following compounds are oxidised 226 according to BIOLOG system: D-trehalose, sucrose, turanose, D-glucose, D-fructose, 227 D-galactose, D-maltose, D-fucose, inosine,sodium lactate, D-arabitol, glycerol, L- 228 glutamic acid, L-serine, pectin, D-galacturonic acid, L-galactonic acid-γ-lactone, D- 229 gluconic acid, D-glucuronic acid, glucuronamide, mucic acid, bromo-succinic acid, 230 acetoacetic acid, D-saccharic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, citric 231 acid, D-malic acid, L-malic acid, γ-amino-n-butyric acid, sodium formate, L- 232 pyroglutamic acid and butyric acid. Resistant to rifamycin SV, vancomycin, tetrazolium 233 violet, tetrazolium blue, niaproof 4, lithium chloride, potassium tellurite and sodium

234 bromate. The major fatty acids consist of C16:0, C16:1ω7c and/or C16:1ω6c and C18:1ω7c. 235 The major respiratory quinone is Q9. The major polar lipids are diphosphatidylglycerol, 236 phosphatidylglycerol and phosphatidylethanolamine.

237 The type strain, EAod3T (=CECT 9073T=LMG 29856T), was isolated from the aerial 238 part of Arthrocnemum macrostachyum. The GenBank/EMBL/DDBJ accession number 239 for the 16S rRNA gene sequence is KU320856. The GenBank/EMBL/DDBJ accession 240 number for the draft genome is ONZI01. The whole genome has a total length of 241 3,764,595 bp and is formed for 64 contigs. The N50 value is 865,511 and the coverage 242 is of 154.703. The genomic G+C content is 59.3%.

243 DESCRIPTION OF KUSHNERIA ENDOPHYTICA SP. NOV.

244 Kushneria endophytica (en.do.phy′ti.ca. Gr. pref. endo within; Gr. n. phyton plant; L. fem. 245 suff. -ica adjectival suffix used with the sense of belonging to; N.L. fem. adj. endophytica 246 within plant, endophytic, referred to the bacterium was found colonising plants 247 endophytically). 248 Cells are Gram-staining-negative, motile, aerobic, 0.1x0.2-0.3 µm in size, and non- 249 spore forming rods frequently appearing as single short rods. Bacteria colonies are light 250 salmon in colour (RAL 060 80 40), smooth, opaque, convex, and viscous, with an 251 undulate margin and 2.6 mm in size when growing on TSA supplemented with 2.5% 252 NaCl (w/v), pH around 7.0 at 30ºC for 2 days. Growth occurs at temperatures of 4-37ºC 253 (optimum 30ºC), although the growth at 4ºC is very weak. The pH range is 5.0-8.0 254 (optimum 7.0). Tolerates until 25% NaCl (w/v) and needs, at least, 0.5% NaCl (w/v), so 255 it is a moderately halophilic microorganism. Does not growth on cetrimide agar. Grows 256 on MacConkey agar and MA. Catalase positive and oxidase negative. Starch and DNA 257 are hydrolysed while casein, Tween 80, chitin, cellulose and pectin are not. Strong 258 enzymatic activity for alkaline phosphatase, esterase lipase (C 8), leucine arylamidase, 259 acid phosphatise,α-glucosidase, β-glucosidase and leucine aminopeptidase; weak 260 enzymatic activity for esterase (C 4), lipase (C 14),α-chymotrypsin, naphthol-AS-BI- 261 phosphohydrolase, α-galactosidase and β-galactosidase (p-nitrophenyl β- 262 Dgalactopyranosidase); and negative for valine arylamidase, cysteine arylamidase, 263 trypsine, β-glucoronidase, N-acetyl-β-glucosaminadase, α-mannosidase, α-fucosidase, 264 arginine dihydrolase, urease, pyrrolidonyl arilamidase (PYR) and β-glucuronidase. 265 Positive for the hydrolysis of esculin; negative for the hydrolysis of gelatine, indole 266 production and production of acetone (Voges-Proskauer negative). Can reduce nitrates 267 to nitrites and assimilate D-mannitol, potassium gluconate and malic acid, but cannot 268 ferment D-glucose or assimilate D-glucose, L-arabinose, D-mannose, N-acetyl- 269 glucosamine, D-maltose, capric acid, adipic acid, trisodium citrate and phenylacetic 270 acid. Acid is produced from D-trehalose, but not from D-ribose, L-arabinose, D- 271 mannitol, D-sorbitol, D-lactose, inulin, D-raffinose, starch and glycogen. The following 272 compounds are oxidised according to BIOLOG system: D-maltose, D-mannose, D- 273 trehalose, sucrose, turanose, D-glucose, D-fructose, D-galactose, D-fucose, inosine, 274 sodium lactate, D-arabitol, glycerol, L-glutamic acid, L-serine, pectin, glycyl-L-proline, 275 D-galacturonic acid, L-galactonic acid-γ-lactone, D-gluconic acid, D-glucuronic acid, 276 glucuronamide, mucic acid, D-saccharic acid, p-hydroxy-phenylacetic acid, methyl 277 pyruvate, citric acid, D-malic acid, L-malic acid, γ-amino-n-butyric acid, sodium 278 formate and butyric acid. Resistant to rifamycin SV, vancomycin, lincomycin, 279 tetrazolium violet, tetrazolium blue, niaproof 4 and sodium bromate. The major fatty

280 acids consist of C16:0, C16:1ω7c/ C16:1ω6c and C18:1ω7c. The major respiratory quinone is 281 Q9. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and 282 phosphatidylethanolamine.

283 The type strain, EAod7T (=CECT 9075T=LMG 29858T), was isolated from the aerial 284 part of Arthrocnemum macrostachyum. The GenBank/EMBL/DDBJ accession number 285 for the 16S rRNA gene sequence is KU320860.

286 287 Acknowledgments

288 This work has been possible thanks to Junta de Andalucía (P11-RNM-7274MO 289 project), INIA (RTA 2012-0006-C03-03 project) and CGL2016-75550-R AEI/FEDER, 290 UE project. S. Navarro-Torre thanks Junta de Andalucía for personal support. LC 291 acknowledges Newcastle University for a postdoctoral fellowship. Genome sequencing 292 of strain EAod3T was provided by MicrobesNG (http://www.microbesng.uk), which is 293 supported by the BBSRC (grant number BB/L024209/1). 294

295 Conflicts of interest

296 The authors declare that there are no conflicts of interest.

297

298 References 299 1. Franzmann PD, Wehmeyer U, Stackebrandt E. Halomonadaceae fam. nov., a 300 new family of the class Proteobacteria to accommodate the genera Halomonas 301 and Deleya. Syst Appl Microbiol 1988; 11:16–19. 302 2. Sánchez-Porro C, de la Haba RR, Soto-Ramírez N, Márquez MC, 303 Montalvo-Rodríguez R, et al. Description of Kushneria aurantia gen. nov., sp. 304 nov., a novel member of the family Halomonadaceae, and a proposal for 305 reclassification of Halomonas marisflavi as Kushneria marisflavi comb. nov., of 306 Halomonas indalinina as Kushneria indalinina comb. nov. and of Halomonas 307 avicenniae as Kushneria avicenniae comb. nov. Int J Syst Evol Microbiol 2009; 308 59(2):397–405. 309 3. Soto-Ramírez N, Sánchez-Porro C, Rosas S, González W, Quiñones M, et al. 310 Halomonas avicenniae sp. nov., isolated from the salty leaves of the black 311 mangrove Avicennia germinans in Puerto Rico. Int J Syst Evol Microbiol 2007; 312 57: 900–905. 313 4. Cabrera A, Aguilera M, Fuentes S, Incerti C, Russell NJ, et al. Halomonas 314 indalinina sp. nov., a moderately halophilic bacterium isolated from a solar 315 saltern in Cabo de Gata, Almería, southern Spain. Int J Syst Evol Microbiol 316 2007; 57: 376–380. 317 5. Yoon J-H, Choi SH, Lee K-C, Kho YH, Kang KH, et al. Halomonas 318 marisflavae sp. nov., a halophilic bacterium isolated from the Yellow Sea in 319 Korea. Int J Syst Evol Microbiol 2001; 51:1171–1177. 320 6. LPSN. 2018. http://www.bacterio.net/kushneria.html. List of prokaryotes 321 standing in nomenclature: genus, Kushneria. Accessed 5 February 2018.

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423 Fig. 1. Total lipid profiles (labelled by the Rf values) of strains EAod3T (a) and EAod7T 424 (b) after separation by two-dimensional TLC using the solvents 425 chloroform/methanol/water (65:25:4, by vol.) in the first dimension, and 426 chloroform/methanol/acetic acid/water (80:12:15:4, by vol.) in the second dimension. 427 Plates were sprayed with molybdatophosphoric acid (3.5 %; Merck) for detection of 428 total polar lipids. PE, Phosphatidylethanolamine; DPG, diphosphatidylglycerol; PG, 429 phosphatidylglycerol; AGL, unidentified aminoglycolipid; PL, unidentified 430 phospholipids. 431 432 Fig. 2. Maximum-likelihood phylogenetic tree inferred from 16S rRNA gene sequences, 433 showing the phylogenetic position of strains EAod3T and EAod7T relative to type 434 strains of species within the family Halomonadaceae. The branches are scaled in terms 435 of the expected number of substitutions per site. Support values obtained from 1000 436 replicates from maximum-likelihood (left) and maximum-parsimony (right) 437 bootstrapping are shown above the branches if ≥60%. Sequence accession numbers are 438 given in parentheses.

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 Table 1. Differential phenotypic characteristics of strains EAod3T, EAod7T and the type strains of the genus Kushneria. Strains: 1, Kushneria sp. nov. EAod3T; 2, Kushneria sp. nov. EAod7T; 3, Kushneria marisflavi DSM 15357T; 4, K. indalinina CECT 5902T; 5, K. pakistanensis KCTC 42082T. +, positive; -, negative; w, weak.

Characteristic 1 2 3 4 5 Cell morphology Rods Rods Rods*a Rods*b Rods*c Colony pigmentation Light Salmon Light Salmon Yellow*a Orange*b Orange*c NaCl for growth (%, w/v) 0.5-25 0.5-25 0.5-27*a 3-25*b 1-30*c Range Optimum 2.5 2.5 0.5-12*a 7.5-10*b 3-9*c pH range 5-8 5-8 5-10*a 5-9*b 6-10.5*c Temperature range (ºC) 4-37 4-37 4-37*a 15-40*b 10-40*c Reduction of nitrate + + -*a +*b +*c Enzyme activity Esterase (C4) + w+ +*c -*c +*c Esterase lipase (C8) + + w+*c -*c w+*c Cystein arylamidase - - w+*c -*c -*c Trypsin - - w+*c -*c -*c Naphtol-As-BI-phosphohydrolase w+ w+ -*c -*c w+*c α-galactosidase - w+ -*c w+*c -*c β-galactosidase w+ w+ -*c +*c +*c β-glucuronidase - - w+*c -*c -*c Hydrolysis of: Aesculin + + +*a -*b +*c Gelatin - - +*a +*b +*c Acid production from: L-Arabinose - - +*a +*b +*c D-Mannitol - - +*a -*b -*c Trehalose + + +*a -*b +*c Growth on: D- Mannitol - + +*a +*b -*c Oxidation of: D- maltose + + - - + L-pyroglutamic acid - - + - + Acetoacetic acid + - - - - Acetic acid - - - + - Glycerol + + + + -

*a Data from Yoon et al. [5]. *b Data from Cabrera et al. [4]. *c Data from Bangash et al. [9]. 455 456 457 458 459 460 461 462 463 464 Table 2. Cellular fatty acid compositions (%) of strains EAod3T, EAod7T and closely related Kushneria species. Strains: 1, Kushneria sp. nov. EAod3T; 2, Kushneria sp. nov. EAod7T; 3, Kushneria marisflavi DSM 15357T; 4, K. indalinina CECT 5902T; 5, K. pakistanensis KCTC 42082T. -, Not detected; tr, values below 1%. All data are from this study. Fatty acid 1 2 3 4 5 C9:0 - tr tr - tr C10:0 - tr 2.0 tr tr C12:0 2.3 2.1 1.1 1.8 1.2 C12:0 2OH 1.4 1.4 2.7 1.8 3.3 C12:0 3OH 9.2 8.7 8.4 8.1 9.4 C14:0 tr tr tr tr tr C16:0 anteiso tr tr tr tr tr Summed feature* 3a 13.7 13.0 7.6 11.3 9.1 C16:0 26.4 27.0 30.7 22.7 23.7 C17:0 anteiso tr tr tr tr tr C17:0 cyclo - - 1.1 tr - Summed feature* 5b - tr tr - tr Summed feature* 8c 44.8 44.6 39.0 49.9 49.3 C18:1 ω5c - - tr - - C18:1 ω7c 11-methyl - - tr - - C18:0 tr tr 1.6 tr tr C19:0 cyclo ω9c - - 3.8 1.5 -

C20:1ω9c tr tr tr tr tr

*Summed features are groups of 2 or 3 fatty acids that are treated together for the purpose of evaluation in the MIDISystem and include both peaks with discrete equivalent chain-lengths (ECLs) as well as those where the ECLs are not reported separately [39]. a Summed feature 3 was listed as C16:1 ω7c and/or C16:1 ω6c. b Summed feature 5 was listed as C18:0 anteiso and/or C18:2 ω6,9c. c Summed feature 8 was listed as C18:1 ω7c and/or C18:1 ω6c.

465 Figure 1 Click here to download Figure Figure 1.eps Figure 2 pacifica KMM 3879T (AB646233) Click here to download Figure Figure 2_final.pdf

Halomonas xinjiangensis TRM 0175 T (EU822512) -/96 Halomonas zincidurans B6 T (JQ781698)

Halomonas anticariensis FP35T (AY489405)

Halomonas socia NY-011T (JF766572) 69/- Halomonas rifensis HK31T (HM026177)

Halomonas desiderata FB2T (X92417)

Halomonas muralis LMG 20969T (AJ320530)

Salinicola peritrichatus DY22T (KC005304)

Salinicola halophilus CG 4.1T (AJ427626)

Chromohalobacter israelensis ATCC 43985T (AJ295144) 100/100 salexigens DSM 3043T (AJ295146) 70/- 99/100 Chromohalobacter canadensis ATCC 43984T (AJ295143)

Halomonas mongoliensis Z-7009T (AY962236) 70/98 Halomomas campaniensis 5AGT (AJ515365)

Halomonas daqiaonensis YCSA28T (FJ984862)

Halomonas fontilapidosi 5CRT (EU541349)

Halomonas alimentaria YKJ-16T (AF211860)

Halomonas ventosae Al12T (AY268080)

Halomonas stenophila N12T (HM242216) 100/100 73/- Halomonas nitroreducens 11ST (EF613113)

Halomonas salifodiane BC7T (EF527873)

Kushneria marisflavi SW32T (AF251143)

Kushneria aurantia A10T (AM941746) 100/100 Kushneria sinocarnis Z35T (FJ667549) 98/66 Kushneria konosiri X49T (GU198748)

Kushneria phyllosphaerae EAod3T (KU320856) 100/100 Kushneria endophytica EAod7T (KU320860)

Kushneria avicenniae MW2aT (DQ888315) 100/100 Kushneria indalinina CG2.1T (AJ427627)

Kushneria pakistanensis NCCP-934T (AB970675)

Zymobacter palmae ATCC 51623T (AF211871) 100/100 nigrificans CTCBS1T (Y13299)

0.03 Supplementary Material Files Click here to download Supplementary Material Files Supplementary Material_v5.pdf

1 Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting

2 endophytes isolated from the halophyte plant Arthrocnemum macrostachyum.

3

4 Salvadora Navarro-Torre1, Lorena Carro2, Ignacio D. Rodríguez-Llorente1, Eloísa Pajuelo1, Miguel Ángel Caviedes1, José

5 Mariano Igual3, Susana Redondo-Gómez4, Maria Camacho5, Hans-Peter Klenk2 & Maria del Carmen Montero-Calasanz2*

6 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Calle Profesor García González, 2, 41012 7 Sevilla, Spain.

8 2School of Natural and Environmental Sciences (SNES), Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.

9 3Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas (IRNASA-CSIC), c/Cordel de 10 Merinas 40-52, 37008 Salamanca, Spain.

11 4Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 1095, 41012 Sevilla, Spain.

12 5IFAPA-Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres-Tomejil, Ctra. Sevilla-Cazalla de la Sierra, Km 12.2, 13 41200 Alcalá del Río, Sevilla, Spain.

14 *Corresponding author: María del Carmen Montero-Calasanz, Tel.: +44 (0)191.208.4943 e-mail: [email protected]

15 Running title: Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov. Supplementary Fig. S1. Optical microscopy photography of strains EAod3T (a) and EAod7T (b) after 48 hours at 30 ºC on TSA plates with 2.5% NaCl (w/v) and pH 7, using the objective 100X.

(a) (b)

10 µm 10 µm

Supplementary Fig. S2. The parameter "Maximum Height" estimated from the respiration curves as measured with the OmniLog phenotyping device and discretized and visualized as heatmap using the opm package. Plates and substrates are rearranged according to their overall similarity (as depicted using the row and column dendrograms). Ochre colour indicates positive reaction; purple colour indicates negative reaction; green colour indicates ambiguous reaction. Letters (A/B) indicate each experimental replicate.

Supplementary Table S1. Genome statistics.

Attribute Value Genome size (bp)a 3,764,595 DNA G + C (%)a 59.3 Number of contigsa 64 Largest contigb 1,327,586 DNA scaffolds 0 Number of CDSc 3419 RNA genesc 68 tmRNAc 1 rRNAc 3 tRNAc 64 N50a 865,511 L50a 2 N75b 699,047 L75b 3 Genes with signal peptidesd 139 Genes with transmembrane helicese 407 CRISPR repeatsf 0 Total number of chromosomes and plasmids 0 a Data from RAST v2.0 [18]. b Data from QUAST v.4.6.3 software [19]. c Data from PROKKA [20]. d Data from SignalP 4.1 server [21]. e Data from TMHMM server v.2.0 [22]. f Data from CRISPRFinder [23].

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