Rossellomorea arthrocnemi sp. nov., a novel plant growthpromoting bacterium used in heavy metal polluted soils as a phytoremediation tool

1 Rossellomorea arthrocnemi sp. nov., a novel plant growthpromoting bacterium used in heavy metal polluted soils as a phytoremediationtool SalvadoraNavarroTorre1, LorenaCarro2, José MarianoIgual3,4 and Maria del CarmenMonteroCalasanz5,* TAXONOMIC DESCRIPTION NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 DOI 10.1099/ijsem.0.005015 Author affiliations: 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Calle Profesor García González, 2, 41012 Sevilla, Spain; 2Microbiology and Genetics Department, University of Salamanca, Salamanca, Spain; 3Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas (IRNASACSIC), c/Cordel de Merinas 4052, 37008 Salamanca, Spain; 4Unidad Asociada Grupo de Interacción PlantaMicroorganismo (Universidad de SalamancaIRNASACSIC), Salamanca, Spain; 5School of Natural and Environmental Sciences (SNES), Newcastle University, Newcastle upon Tyne, NE1 7RU, UK. *Correspondence: Maria del Carmen MonteroCalasanz, maria. monterocalasanz@ ncl. ac. uk Keywords: Arthrocnemum macrostachyum; Odiel marshes; plant growth; PGPR; endophyte. Abbreviations: ANI, average nucleotide identity; dDDH, digital DNADNA hybridization; IAA, indole3acetic acid; MA, marine agar; MK, menaquinone; PGP, plant growthpromoting; TLC, thinlayer chromatography; TSA, tryptic soy agar; TYGS, type genome server. The GenBank/EMBL/DDBJ accession number for the complete 16S rRNA gene sequence is MZ416782. The GenBank/EMBL/DDBJ accession number for the draft genome is CAJGBF010000000. Three supplementary tables and three supplementary figures are available with the online version of this article. 005015 © 2021 The Authors This is an openaccess article distributed under the terms of the Creative Commons Attribution NonCommercial License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution. Abstract Strain EAR8T is a root endophyte isolated from Arthrocnemum macrostachyum plants collected from the Odiel marshes, Huelva (Spain). It presented in vitro plant growthpromoting properties and improved the plant growth and heavy metal accumulation in polluted soils playing an important role in phytoremediation strategies. Phenotypically, strain EAR8T cells were Grampositive, aerobic and nonmotile rods with terminal oval endospores and nonswollen sporangia which form beige, opaque, butyrous, raised and irregular colonies with undulate margins. The strain was able to grow between 15–45 °C, at pH 6.0–9.0 and tolerated 0–25 % NaCl (w/v) showing optimal growth conditions on trypticase soy agar plates supplemented with 2.5 % NaCl (w/v) at pH 7.0 and 37 °C for 24 h. Chemotaxonomic analyses showed that the isolate has mesodiaminopimelic acid as the peptidoglycan in the cell wall and MK7 as the major respiratory quinone. The predominant fatty acids were anteisoC15 : 0 and isoC15 : 0 and the polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on the whole proteomes of closest sequenced relatives confirmed that strain EAR8T is affiliated to the genus Rossellomorea and forms a clade with Rossellomorea vietnamensis 151T with maximum support. Genome analyses showed that EAR8T has indole3acetic acid and siderophore biosynthesis and transporters genes and genes related to resistance against heavy metals. Phenotypic and phylogenomic comparative studies suggested that strain EAR8T is a new representative of the genus Rossellomorea and the name Rossellomorea arthrocnemi sp. nov. is proposed. Type strain is EAR8T (=CECT 9072T=DSM 103900T). The genus Rossellomorea, belonging to the family Bacillaceae (phylum Firmicutes), is a very recenty genus proposed by Gupta et al. [1] to encompass previously described Bacillus species that can reliably be distinguished from all other Bacillaceae species by the presence of exclusively shared conserved signature indels in their protein sequences. At the time of writing, the genus comprises up to four validly named species [2], with Rossellomorea aquimaris as the type species. Species from genus Rossellomorea have mainly been isolated from different saline environments [3–5]. No representative of Rossellomorea have been isolated from plants so far. Strain EAR8T is an endophytic bacterium isolated from roots of Arthrocnemum macrostachyum, a halophyte plant growing in the Odiel marshes, Huelva, Spain [6]. Previous studies showed that EAR8 T is an efficient plant growthpromoting (PGP) bacterium able to produce siderophores and synthetise auxins [6]. This strain also tolerates up to 15 mM Ni, 9 mM Pb, 8 mM As and 2 mM Cd, among others [6]. Moreover, it was part of a bacterial consortium that improved seed germination, growth and heavy metal accumulation in roots of A. macrostachyum plants in heavy metal polluted soils, enhancing the potential of this halophyte to be used as a phytoremediation tool [6, 7]. OPEN ACCESS 2 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 Based on the demonstrated efficiency of this PGP strain for bioaugmentation processes to recover heavy metal contaminated soils, it is worthwhile to determine its exact phylogenetic position. This study therefore aims to characterize plant endophyte strain EAR8T as a potential novel species in the genus Rossellomorea by using a polyphasic approach. ISOLATION AND ECOLOGY Strain EAR8T was isolated from roots of A. macrostachyum from the Odiel marshes, Huelva, Spain (37° 13′ N, 6° 57′ E) as described by NavarroTorre et al. [6]. Briefly, after root surface disinfection, endophyte extraction was performed in 0.9 % sterile saline (w/v) solution using a sterile mortar. The extract was plated on tryptic soy agar (TSA) plates supplemented with 0.3 M NaCl and incubated at 28 °C for 72 h. Isolates were identified and classified according to their colony characteristics and pure cultures were stored in 15 % glycerol at −80 °C for longterm preservation. 16S RNA PHYLOGENY Genomic DNA was extracted using an igenomic BYF DNA Extraction kit (Intron Biotechnology) according to the manufacturer’s instructions. The 16S rRNA gene was then amplified by PCR using the primers and conditions described by NavarroTorre et al. [6] and the product was sequenced by StabVida (Portugal). 16S rRNA gene partial sequence was deposited in the GenBank/EMBL/DDBJ database under the accession number KU320872 (complete 16S rRNA gene sequence as extracted from the genome sequence can be found under accession number MZ416782). This sequence was aligned with the sequences of the closely related type strains using the EzTaxone service ( www. ezbiocloud. net/ eztaxon) [8] and pairwise similarities were determined as described by MeierKolthoff et al. [9]. A phylogenetic tree was created using the GGCD web server (http:// ggdc. dsmz. de/) [10] according to MonteroCalasanz et al. [11]. The 16S rRNA gene sequence of strain EAR8T confirmed that it belonged to the genus Rossellomorea showing the highest sequence similarities to Rossellomorea aquimaris TF12T (97.4 %) and Rossellomorea vietnamensis 151T (97.2 %). These similarities were, nevertheless, lower than the threshold recommended (98.8–98.7 %) to determine a new species within the phylum Firmicutes [9, 12], indicating the unambiguous novelty of strain EAR8T within the genus Rossellomorea. Phylogenetic inferences also confirmed its affiliation by placing the isolate in a wellsupported clade within the genus Rossellomorea (Fig.1). GENOME FEATURES The whole genome of strain EAR8T was sequenced by MicrobesNG (Birmingham, UK) using Illumina technology. Kraken was used to identify the closest available reference [13]. The quality of data was studied by mapping the reads with BWA mem [14], de novo assembly of the genome was performed using SPAdes [15] and, then, more quality parameters were checked with BWA mem. The whole genome was deposited in GenBank/EMBL/DDBJ under the accession number CAJGBF010000000. Genome annotation was performed with Prokka [16] and basic statistics about the genome were extracted using the rast server version 2.0 [17], quast version 4.6.3 software [18], the SignalP 4.1 server [19], the tmhmm server v.2.0 [20], CRISPRFinder [21] and PlasFlow [22]. According to the genome sequence analyses, the draft genome sequence of strain EAR8T had a total length of 4 775 586 bp and was formed of 107 contigs with a coverage of 80.6×. The N50 value was 199 119 and the G+C content 42 mol% (Table S1). A phylogenetic tree based on whole proteomes was inferred using the Type (Strain) Genome Server (TYGS; https:// tygs. dsmz. de/) [23]. The phylogenetic inference was performed via FastME 2.1.4 including SPR postprocessing [24]. The tree was rooted at the midpoint [25] and visualized with PhyD3 [26]. The phylogenetic tree based on whole proteogenomes confirmed the affiliation within the genus Rossellomorea with maximum support for R. vietnamensis NBRC 101237T as the closest species (Fig.2). Finally, taxogenomic analyses were carried out with the closely related species calculating the overall genome related indexes. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) tests were determined using the TYGS web server (https:// tygs. dsmz. de/) [23] and JSpeciesWS server (http:// jspecies. ribohost. com/ jspeciesws) [27], respectively. dDDH results showed 28.7 and 20.3% similarities with genomes of R. vietnamensis NBRC 101237T (accession number BCVQ00000000) and R. aquimaris TF12T (accession number LQXM00000000), respectively. Furthermore, ANI tests reported 84.1 % (ANIb) and 86.3 % (ANIm) with R. vietnamensis NBRC 101237T and 75.6 % (ANIb) and 83.7 % (ANIm) relatedness with R. aquimaris TF12T. Accordingly, all values were well below the recommended thresholds for species delineation [9, 28]. As mentioned above, strain EAR8T has some PGP properties such as siderophore and auxin production [6]. Using the rast server version 2.0 [17], genes involved in these properties were searched for in the genome. Regarding the production of siderophores, genes related to the synthesis of different siderophores were annotated. In fact, all genes involved in pyochelin biosynthesis (pchR, pchA, pchB, pchC, pchD, pchE, pchF and pchG) described in Pseudomonas aeruginosa [29] were located into the EAR8T genome (Table S2). In addition, genes encoding the enzymes isochorismatase and 2,3dihydr o2,3dihydroxybenzoate dehydrogenase, key in the synthesis of bacillibactin, a siderophore produced by members of family Bacillaceae [30, 31], were identified. Moreover, other enzymes involved in the synthesis of enterobactin, vibriobactin, rhizobactin 1021, baumannoferrin and aerobactin were also observed. Finally, genes related to siderophore transport systems, such as permeases and ATPases components, and receptors were also present in the genome (Table S2). 3 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 Regarding auxin production, the EAR8T genome showed genes involved in tryptophan biosynthesis and indole3acetic acid (IAA) biosynthesis. Tryptophan is the main precursor to IAA synthesis. It is involved in five different wellstudied pathways. That is, the indol3acetamide pathway, the indole3pyruvate pathway, the tryptamine pathway, the tryptophan sidechain oxidase pathway and the indole3acetonitrile pathway [32]. The tryptamine and indole3acetonitrile pathways have been described as the main IAA routes in the closer genus Bacillus [33, 34]. Both pathways were also identified in the EAR8T´s genome sequence in addition to complete sets of genes involved in the indol3acetamide and indole3pyruvate pathways (Table S2). It supported the in vitro IAA production observed for this strain in previous works [6]. Finally, the presence of genes related to heavy metal tolerance (Table S3) confirmed the resistance to As, Cu, Pb, Zn, Cd, Hg and Ni previously observed [6]. PHYSIOLOGY AND CHEMOTAXONOMY Growth conditions were studied on TSA plates supplemented with 0.3 M NaCl at different temperatures (4, 15, 20, 25, 28, 30, 32, 37 and 45 °C) and at different pH values (pH 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0; at 28 °C) for 6 days. pH values were adjusted using a citrate–phosphate buffer (0.1 M citric acid and 0.2 M dibasic sodium phosphate) and a Tris–HCl buffer [0.1 M Tris (hydroxymethyl) aminomethane and 0.1 M HCl]. NaCl tolerance was examined by incubating the isolate on membrane tryptone–glucose extract agar plates with varying NaCl concentrations (0, 0.5, 2.5, 5, 7.5, 10, 12.5, 17.5, 20, 25 Fig. 1. Maximumlikelihood phylogenetic tree inferred from 16S rRNA gene sequences, showing the phylogenetic position of strain EAR8T relative to type strains of species within the genus Rossellomorea. The branches are scaled in terms of the expected number of substitutions per site. Support values obtained from 1000 replicates from maximumlikelihood (left) and maximumparsimony (right) bootstrapping are shown above the branches if≥60 %. Sequence accession numbers are given in parentheses. 4 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 and 30 %; w/v) at 28 °C for 6 days [35]. Anaerobic respiration was tested in tubes with semisolid TSA supplemented with 2.5 % NaCl (w/v) covered with 2 ml agar (2 % w/v) and, then, with 2 ml paraffin. Tubes were incubated at 28 °C for 10 days [36]. Additional cultural features were tested on marine agar (MA) and two selective media, cetrimide agar and MacConkey agar, both supplemented with 2.5 % NaCl (w/v). Colony characteristics were observed on TSA plates supplemented with 2.5 % NaCl (w/v) grown at 37 °C after 24 h using a stereoscopic microscope (Olympus SZ61). Colony colour was determined by the colour chart RAL D2 Design. Cell morphology was examined in the growth phase using an optical microscope with a ×100 objective (Olympus CX41) after Gram staining [37]. Finally, cell motility was studied by observing a drop of liquid culture under optical microscopy with a ×40 objective [6]. Cells of strain EAR8T were Gramstainpositive, aerobic and nonmotile rods of 0.3–0.4×1.4–2 µm (in growth phase) occurring singly. Terminal oval endospores and nonswollen sporangia were also identified (Fig. S1) in concordance with related species [3, 4, 38] and in line with the genus description [1]. However, the absence of motility is a trait only shared with a few species of the closer genus Bacillus i.e. Bacillus anthracis, Bacillus megaterium and Bacillus mycoides [39], being the first nonmotile representative in Rossellomorea. After 24 h at 37 °C on TSA 2.5 % NaCl (w/v) plates, cells formed beige (RAL 090 90 10), opaque, butyrous, raised and irregular colonies with an undulate margin whose diameter was 3.8 mm. After 7 days, a curled margin appeared. Growth was observed at pH from pH 6.0 to 9.0 (optimum pH at 7.0–8.0) and at temperature from 15 to 45 °C (optimum temperature at 37 °C). The temperature range for growth was similar to those shown by the reference strains (Table1). Regarding NaCl tolerance, strain EAR8T tolerated up to 25 % NaCl (w/v) and it was able to grow in absence of NaCl, so it could be considered an extremely halotolerant bacterium according to the classification suggested by Ventosa et al. [40]. Closely related species were also described as halotolerant [3–5, 38], but strain EAR8T was shown to be the most salt tolerant strain in this study (Table1). Oxidase activity was determined adding 1 % N,N,N′,N′- tetramethylpphenylenediamine powder (Becton, Dickinson and Company) to the bacterial biomass. Oxidase test was considered positive if the colour of the biomass turned blue. For the catalase activity, a drop of 3 % H2O2 was added to the bacterial biomass. The presence of bubbles indicated if the test was positive. Strain EAR8T was catalasepositive but oxidasenegative (Table1) in correlation with previous descriptions for most species of the genus. Biochemical characterizations of strain EAR8T were performed using the API 20NE, API 20Strep and API ZYM galleries (bioMérieux) according to the manufacturer’s instructions. In addition, GEN III MicroPlates (Biolog) were Fig. 2. Phylogenomic tree inferred with GBDP from whole proteomes showing the phylogenetic position of strain EAR8T relative to closest species. The numbers above branches are GBDP pseudobootstrap support values from 100 replications. 5 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 Table 1. Differential characteristics among strain EAR8T and the closely related type strains of the genus Rossellomorea. Strains: 1, Strain EAR8T; 2, R. aquimaris TF12T; 3, R. vietnamensis 151T; 4, R. marisflavi TF11T; 5, R. oryzaecorticis R1T. +, positive; −, negative; w, weak; nd, no data available. Characteristics 1 2 3 4 5 NaCl range for growth (%, w/v) 0–25 0–18a0–15c0–16a0–9b Temperature range for growth (°C) 15–45 10–44a10–40c10–47a15–45b Optimum temperature for growth (°C) 37 30–37a31–40c30–37a37b Oxidase activity − −a+c−a+b Hydrolysis of: Aesculin + −a+c+and Gelatin + −b+c+b−b Urease activity + −a−c−a−b Assimilation of: lArabinose +−b−c−b+b dmannose +−b−c+b+b dmannitol + −b+c+b+b NAcetylglucosamine + −b+cwb−b Potassium gluconate − nd +cnd +b Acid production from: dRibose − +a+c+awd dMannitol − −a+c+a+d Lactose + −a−c−a−d dRaffinose − −a+cwa−d Glycogen − +a+c−a+d Oxidation of: βGentiobiose − − + −b−b Turanose − + + −b−b βMethyldglucoside + + − nd −b dSalicin + − − +b−b dMannose + − − nd nd 3OMethyldglucose − + + nd nd dFucose + + − −b−b dArabitol − + + nd −b myoInositol + − − −b−b dGlucuronic acid + − − nd nd dSaccharic acid + − − nd nd Citric acid + − − nd nd lMalic acid + − − nd nd βHydroxybutyric acid + − − nd nd Propionic acid − + + nd nd Continued 6 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 used to test carbon source utilization and chemical sensitivity. the GEN III MicroPlates were inoculated using liquid cultures resuspended in a viscous inoculating fluid C supplemented with 2.5 % NaCl (w/v) at 90–95 % transmittance. Then, those were incubated at 30 °C for 3 days in an Omnilog device (Biolog). Obtained results were analysed with the opm package for R version 1.3.72 [41, 42]. Reference strains R. aquimaris DSM 16205T and R. vietnamensis DSM 18898T were tested in parallel experiments. Previous data from NavarroTorre et al. [6] also indicated that strain EAR8T hydrolyses casein and DNA but not starch, Tween 80, cellulose, chitin and pectin. Additional biochemical results from the API 20NE and API ZYM galleries and the GEN III MicroPlates can be found in Table1 and Fig. S2. A detailed list of them are also provided in the protologue. For chemotaxonomic analysis, respiratory quinones and polar lipids were extracted from freezedried biomass using an aqueous methanol–petroleum ether (1 : 1, v/v) solution following the combined protocol established by Minnikin et al. [43]. Respiratory quinone residue was then dissolved in isopropanol and identified by highperformance liquid chromatography [44]. Recovered polar lipids extracts were analysed by 2D thinlayer chromatography (TLC) [43]. Polar lipid identification was carried out by spraying different reagents over the TLC plates (0.2 % ninhydrin in acetone, α-naphtolsulphuric acid, 1.3 % molybdenum blue, Dragendorff reagent and 5 % molibdatophosphoric acid in ethanol to detect aminogroups, glycogroups, phosphogroups, cholinegroups and all total lipids, respectively) [45, 46]. In addition, 40 mg fresh biomass grown on TSA plates supplemented with 0.3 M NaCl for 24 h at 28 °C wase harvested to extract fatty acids following the protocol outlined by Sasser [47]. Reference strains indicated previously were analysed in parallel experiments using the same growth conditions. The identification of fatty acids was performed using the Microbial Identification System (midi) Sherlock version 6.1 (TSBA40 database). Lastly, Staneck and Roberts’s protocol [48] was used to identify the stereoisomer of diaminopimelic acid in the peptidoglycan of strain EAR8T. Strain EAR8T presented mesodiaminopimelic acid in the cellwall peptidoglycan and MK7 as the major respiratory quinone (88 %), similarly to what was already described for other Rossellomorea representatives. Minor MK components (<10 %) of MK6 (4 %), MK8 (3 %) and MK9 (0.3 %) were also identified. The fatty acid pattern was mainly composed of anteisoC15 : 0 and isoC15 : 0 (Table2) as outlined for other related species used in this study and in correlation with what was already observed in other species [3, 38]. Finally, the polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine (Fig. S3). A similar pattern was observed for other species in the genus such as Rosseallomorea oryzaecorticis [38]. Characteristics 1 2 3 4 5 Resistance to: Nalidixic acid − + + nd nd 1 % Sodium lactate − + + nd nd Sodium formate − + + nd nd a, Data from Yoon et al. [3]. b, Data from Hong et al. [38]. c, Data from Noguchi et al. [4]. d, Data from Daroonpunt et al. [49]. Table 1. Continued Table 2. Cellular fatty acid patterns (%) of strain EAR8T and closely related species Strains: 1, EAR8T; 2, R. aquimaris DSM 16205T; 3, R. vietnamensis DSM 18898T. −, Not detected; tr, values below 1 %. Values below 1 % in all columns are not displayed. All data were obtained in this study. Fatty acid 1 2 3 isoC14 : 0 4.0 1.1 2.2 isoC15 : 0 21.6 19.9 15.0 anteisoC15 : 0 52.2 34.8 36.3 C16 : 1 ω7c alcohol 4.0 – tr isoC16 : 0 2.2 1.9 6.8 C16 : 0 tr 2.4 10.4 Summed feature 4* 3.7 tr tr isoC17 : 0 tr 4.3 11.4 anteisoC17 : 0 5.7 16.5 12.7 C18 : 0 tr 2.9 tr anteisoC19 : 0 – 6.5 – C20 : 0 – 3.4 – *Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The midi system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 4 was listed as isoC17 : 1 and/or anteisoC17 : 1. 7 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 Considering the results of phylogenetic analysis and dDDH and ANI relatedness studies, and on the basis of phenotypic evidence, we propose that strain EAR8 T represents a novel species in the genus Rossellomorea, for which the name Rossellomorea arthrocnemi sp. nov. is proposed. DESCRIPTION OF ROSSELLOMOREA ARTHROCNEMI SP. NOV. Rossellomorea arthrocnemi ( ar. thro. cne’mi. N.L. gen. neut. n. arthrocnemi, of Arthrocnemum macrostachyum, where the type strain was isolated from). Cells are Gramstainpositive, aerobic and nonmotile rods. Endospores are terminal and oval in nonswollen sporangia. Colonies are beige, opaque, butyrous, raised and irregular with undulate margins on TSA 2.5 % NaCl (w/v) plates at 37 °C for 24 h (optimal growth conditions). Growth ranges are pH 6.0–9.0 (optimum at 7.0–8.0) and 15–45 °C (optimum at 37 °C). It tolerates 0–25 % NaCl (w/v) and grows on MA plates but not on cetrimide or MacConkey agar media. Catalasepositive and oxidasenegative. Aesculin, casein, DNA and gelatin are hydrolysed, butnot cellulose, chitin, pectin, starch and Tween 80 are not. Reduces nitrates to nitrites; assimilates dglucose, maltose, dmannitol, dmannose, larabinose, malic acid and Nacetyldglucosamine; ferments dglucose; but cannot assimilate adipic acid, capric acid, phenylacetic acid, potassium gluconate and trisodium citrate. Negative for arginine dihidrolase and indole production and positive for urease activity. Acid is produced from lactose, trehalose and starch, but not from dmannitol, draffinose, dribose, dsorbitol, glycogen, inulin and larabinose. Positive for the Voges–Proskauer test and negative for leucine aminopeptidase and pyrrolidonyl arilamidase activities. Strong enzymatic activity is observed for alkaline phosphatase and βglucosidase; weak activity for acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, naphtholASBIphosphohydrolase and αglucosidase; and no activity for acid phosphatase, cystine arylamidase, lipase (C14), Nacetyl-βglucosaminidase, trypsin, valine arylamidase, αchymotrypsin, αfucosidase, αgalactosidase, αmannosidase, βgalactosidase and βglucuronidase. Acetic acid, acetoacetic acid, citric acid, dextrin, dfructose, dfructose6phosphate, dfucose, dgluconic acid, dglucuronic acid, dglucose, dlactic acid methyl ester, maltose, dmannitol, dmannose, dsaccharic acid, dsalicin, trehalose, gelatin, glycerol, glycyllproline, larginine, laspartic acid, lgalactonic acid-γlactone, lglutamic acid, lhistidine, llactic acid, lmalic acid, lpyroglutamic acid, lserine, methyl pyruvate, myoinositol, Nacetyldglucosamine, Nacetyl-βdmannosamine, pectin, sucrose, Tween 40, αketoglutaric acid, βhydroxybutyric acid and βmethyldglucoside are oxidized but not bromosuccinic acid, butyric acid, darabitol, daspartic acid, cellobiose, dgalactose, dgalacturonic acid, dglucose6phosphate, dmalic acid, melibiose, draffinose, dsorbitol, turanose, serine, glucuronamide, inosine, lalanine, lfucose, lrhamnose, 3methylglucose, mucid acid, Nacetyldgalactosamine, Nacetyldneuraminic acid, phydroxyphenylacetic acid, propionic acid, quinic acid, stachytose, lactose, αhydroxybutyric acid, αketobutyric acid, βgentiobiose and γaminobutyric acid. It tolerates aztreonam, lithium chloride and potassium tellurite, but not fusidic acid, guanidine HCl, lincomycin, minocycline, nalidixic acid, niaproof 4, rifampicin SV, sodium bromate, sodium formate, 1 % sodium lactate, tetrazolium blue, tetrazolium violet, troleandomycin and vancomycin. Mesodiaminopimelic acid is present in the the cellwall peptidoglycan and MK7 is the major respiratory quinone. The predominant fatty acids are anteisoC15 : 0 and isoC15 : 0. The polar lipid profile is composed of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The genome of strain EAR8T has a total length of 4 775 586 bp, formed of 107 contigs and has a coverage of 80.6×. The N50 value is 199 119 and the G+C content is 42 mol%. The type strain, EAR8T (=CECT 9072T=DSM 103900T), was isolated as a root endophyte of the halophyte Arthrocnemum macrostachyum. Funding information This work has been possible thanks to Junta de Andalucía (P11RNM7274MO project) and INIA (RTA 20120006 C0303 project). Thanks to DSMZ for supplying the type reference strains. S. N.-T. also thanks Junta de Andalucía for personal support. L.C. thanks Salamanca University for a postdoctoral fellowship. Acknowledgements Thanks to CECT and DSMZ for accepting and maintaining the studied strain in their bacterial collections. Conflicts of interest The authors declare that there are no conflicts of interest. References 1. Gupta RS, Patel S, Saini N, Chen S. Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species. Int J Syst Evol Microbiol 2020;70:5753–5798. 2. Parte AC, Sardà Carbasse J, MeierKolthoff JP, Reimer LC, Göker M. List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ. Int J Syst Evol Microbiol 2020;70:5607–5612. 3. Yoon JH, Kim IG, Kang KH, TK O, Park YH. Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 2003;53:1297–1303. 4. Noguchi H, Uchino M, Shida O, Takano K, Nakamura LK, et al. Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endosporeforming bacterium isolated from Vietnamese fish sauce. Int J Syst Evol Microbiol 2004;54:2117–2120. DOI: DOI: 10.1099/ijs.0.028950 5. Dastager SG, Mawlankar R, Tang SK, Srinivasan K, Ramana VV, etal. Bacillus enclensis sp. nov., isolated from sediment sample. Antonie vanVan Leeuwenhoek 2014;105:199–206. DOI: DOI: 10.1007/ s1048201300663 6. NavarroTorre S, MateosNaranjo E, Caviedes MA, Pajuelo E, RodríguezLlorente ID. Isolation of plantgrowthpromoting and metalresistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation. Mar Pollut Bull 2016;110:133–142.:S0025326X(16)30474X. 7. NavarroTorre S, BarciaPiedras JM, Caviedes MA, Pajuelo E, RedondoGómez S, etal. Bioaugmentation with bacteria selected 8 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 from the microbiome enhances Arthrocnemum macrostachyum metal accumulation and tolerance. Mar Pollut Bull 2017;117:340–347. 8. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and wholegenome assemblies. Int J Syst Evol Microbiol 2017;67:1613–1617. 9. MeierKolthoff JP, Göker M, Spröer C, Klenk HP. When should a DDH experiment be mandatory in microbial taxonomy. Arch Microbiol 2013;195:413–418. 10. MeierKolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequencebased species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 2013;14:60. 11. MonteroCalasanz MC, Göker M, Pötter G, Rohde M, Spröer C, et al. Geodermatophilus arenarius sp. nov., a xerophilic actinomycete isolated from Saharan desert sand in Chad. Extremophiles 2012;16:903–909. DOI: DOI: 10.1007/s0079201204864 12. Chun J, Oren A, Ventosa A, Christensen H, Ruiz Arahal D, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2018;68:461– 466. DOI: DOI: 10.1099/ijsem.0.002516 13. Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 2014;15:R46. 14. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWAMEM. 2013:arXiv:1303.3997v2. 15. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M. SPAdes: A new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 2012;19:455–477. 16. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014;30:2068–2069. 17. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, et al. The RAST server: rapid annotations using subsystems technology. BMC Genomics 2008;9:75. DOI: DOI: 10.1186/14712164975 18. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 2013;29:1072–1075. 19. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011;8:785–786. 20. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001;305:567–580. 21. Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 2007;35:W52–W57. 22. Krawczyk PS, Lipinski L, Dziembowski A. PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures. Nucleic Acids Res 2018;46:e35. 23. MeierKolthoff JP, Göker M. TYGS is an automated highthroughput platform for stateoftheart genomebased taxonomy. Nat Commun 2019;10:2182. 24. Lefort V, Desper R, Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distancebased phylogeny inference program. Mol Biol Evol 2015;32:2798–2800. 25. Farris JS. Estimating phylogenetic trees from distance matrices. Am Nat 1972;106:645–667. 26. Kreft L, Botzki A, Coppens F, Vandepoele K, Van Bel M. PhyD3: A phylogenetic tree viewer with extended phyloXML support for functional genomics data visualization. Bioinformatics 2017;33:2946–2947. 27. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 2016;32:929–931. 28. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 2009;106:19126–19131. 29. Marathe R, Phatake Y, Sonawane A. Bioprospecting of Pseudomonas aeruginosa for their potential to produce siderophore: process optimization and evaluation of its bioactivity. Int J Bioassays 2015;4:3667–3675. 30. Hotta K, Kim CY, Fox DT, Koppisch AT. Siderophoremediated iron acquisition in Bacillus anthracis and related strains. Microbiology 2010;156:1918–1925. 31. May JJ, Wendrich TM, Marahiel MA. The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3dihydroxybenzoateglycinethreonine trimeric ester bacillibactin. J Biol Chem 2001;276:7209–7217. 32. Duca DR, Glick BR. Indole3acetic acid biosynthesis and its regulation in plantassociated bacteria. Appl Microbiol Biotechnol 2020;104:8607–8619. 33. Perley JW, Stowe BB. On the ability of Taphrina deformans to produce indoleacetic acid from tryptophan by way of tryptamine. Plant Physiol 1966;41:234–237. 34. Kato Y, Nakamura K, Sakiyama H, Mayhew SG, Asano Y. Novel hemecontaining lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB1: Purification, characterization, and molecular cloning of the gene. Biochemistry 2000;39:800–809. 35. Bangash A, Iftikhar A, Saira A, Takuji K, Armghan S, etal. Kushneria pakistanensis sp. nov., a novel moderately halophilic bacterium isolated from rhizosphere of a plant (Saccharum spontaneum) growing in salt mines of the Karak area in Pakistan. Antonie van Leeuwenhoek 2015;107:991–1000. DOI: DOI: 10.1007/ s1048201503919 36. Zou Z, Wang G. Kushneria sinocarnis sp. nov., a moderately halophilic bacterium isolated from a Chinese traditional cured meat. Int J Syst Evol Microbiol 2010;60:1881–1886. 37. Halebian S, Harris B, Finegold SM, Rolfe RD. Rapid method that aids in distinguishing Grampositive from Gramnegative anaerobic bacteria. J Clin Microbiol 1981;13:444–448. 38. Hong SW, Kwon SW, Kim SJ, Kim SY, Kim JJ, etal. Bacillus oryzaecorticis sp. nov., a moderately halophilic bacterium isolated from rice husks. Int J Syst Evol Microbiol 2014;64:2786–2791. DOI: DOI: 10.1099/ijs.0.0587680 39. Logan NA, Vos PD. Bacillus. Whitman WB, Rainey F, Kämpfer P, Trujillo M and Chun J (eds). In: Bergey’s Manual of Systematics of Archaea and Bacteria. John Wiley & Sons, Inc., in association with Bergey’s Manual Trust; 2015. 40. Ventosa A, Nieto JJ, Oren A. Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 1998;62:504–544. 41. Vaas LAI, Sikorski J, Michael V, Göker M, Klenk HP. Visualization and curve parameter estimation strategies for efficient exploration of phenotype microarray kinetics. PLoS One 2012;7:e34846. 42. Vaas LAI, Sikorski J, Hofner B, Fiebig A, Buddruhs N, etal. opm: an R package for analyzing OmniLog (R) phenotype microarray data. Bioinformatics 2013;29:1823–1824. DOI: DOI: 10.1093/ bioinformatics/btt291 43. Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athayle M, etal. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 1984;2:233– 241. DOI: DOI: 10.1016/01677012(84)900186 44. Kroppenstedt RM, Goodfellow M. The family Thermonosporaceae: Actinocorallia, Actinomadura, Spirillispora y Thermomonospora. Dworkin M, Falkow S, Schleifer KH and Stackebrandt E (eds). In: Archaea y Bacteria, 3st ed edn, Vol. 3. New York: Springer; 2006. pp. 682–724. 45. Tindall BJ. A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 1990;13:128–130. 46. Tindall BJ. Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 1990;66:199–202. 47. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. USFCC Newsl 1990;20:16. 9 NavarroTorre etal., Int. J. Syst. Evol. Microbiol. 2021;71:005015 48. Staneck JL, Roberts GD. Simplified approach to identification of aerobic actinomycetes by thinlayer chromatography. Appl Microbiol 1974;28:37. 49. Daroonpunt R, Yiamsombut S, Sitdhipol J, Tanasupawat S. Bacillus salacetis sp. nov., a slightly halophilic bacterium from Thai shrimp paste (Kapi). Int J Syst Evol Microbiol 2019;69:1162–1168. Five reasons to publish your next article with a Microbiology Society journal 1. The Microbiology Society is a not-for-profit organization. 2. We offer fast and rigorous peer review – average time to first decision is 4–6 weeks. 3. Our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as ‘excellent’ or ‘very good’. 5. 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