DOI: 10.15468/x5qscx
title: Pontibacter brevis sp. nov., isolated from rhizosphere soil of Tamarix ramosissima

description: This dataset contains the digitized treatments in Plazi based on the original journal article Osman, Ghenijan, Gao, Yan, Wang, Ning, Mahmud, Otkur, Sun, Jian, Zhang, Tao, Zhan, Faqiang, Zhang, Zhidong, Lou, Kai (2018): Pontibacter brevis sp. nov., isolated from rhizosphere soil of Tamarix ramosissima. International Journal of Systematic and Evolutionary Microbiology 68 (1): 81-86, DOI: 10.1099/ijsem.0.002455, URL: http://dx.doi.org/10.1099/ijsem.0.002455AbstractA Gram-stain-negative, oval-shaped and light pink pigmented bacterium, designated XAAS-2 T, was isolated from rhizosphere soil of Tamarix ramosissima. The sole respiratory quinone of the type strain XAAS-2 T was MK-7, and the principal cellular fatty acids were summed feature 4 (iso-C 17: 1 I and/or anteiso-C 17: 1 B) and iso-C 15: 0. The major polar lipids were phosphatidylethanolamine and two unidentified lipids. 16S rRNA gene sequence analysis indicated that strain XAAS-2 T belonged to the genus Pontibacter within the family Cytophagaceae with sequence similarities of 93.9–97.1 % to other type species of the genus Pontibacter and to Pontibacter xinjiangensis CCTCC AB 207200 T as the closest neighbour. The DNA G+C content of strain XAAS-2 T was 50.6 mol%. The level of DNA–DNA relatedness of XAAS-2 T and P. xinjiangensis CCTCC AB 207200 T was 47.5 % (SD=3.27). Phenotypic and genotypic data suggested that strain XAAS-2 T represents a novel species of the genus Pontibacter, for which the name Pontibacter brevis sp. nov. is proposed, with the type strain XAAS-2 T (=CCTCC AB 2016135 T =JCM 31443 T).The genus Pontibacter of the family Cytophagaceae of the phylum Bacteriodetes was first described by Nedashkovskaya et al. [1] with Pontibacter actiniarum as the type species. Members of this genus are aerobic, Gram-stainnegative, rod-shaped, non-spore forming, motile or nonmotile bacteria that possess phosphatidylethanolamine (PE) as the major polar lipid and menaquinone 7 (MK-7) as the major respiratory quinone [1, 2]. At the time of writing, there were 29 species of this genus with validly published names (www.bacterio.net), and these have been isolated from a marine coelenterate, solar saltern, desert sand, sea water, forests, mountains, plant rhizospheres, pond sediments, a hexachlorocyclohexane dumpsite and a salt lake [1 – 10].During a survey of the diversity and phylogeny of culturable bacteria in salt lakes in Xinjiang, China, many bacterial strains were isolated from plant rhizosphere soil and plant roots collected from the shore of Aydingkol Lake near the city of Turpan, Xinjiang, China. One of them, designated XAAS-2 T, was phylogenetically related to the genus Pontibacter. Phylogenetic analysis based on 16S rRNA gene sequence data showed that sequence similarity between XAAS-2 T and related strains of the genus Pontibacter was below 97.1 %. Therefore, we report the isolation and polyphasic taxonomic examination of strain XAAS-2 T.Strain XAAS-2 T was isolated from the rhizosphere soil of Tamarix ramosissima collected from the shore of Aydingkol Lake (GPS position: 89 Ǫ 16′ 30.8″ E, 42 Ǫ 40′ 12.6″ N). The soil sample was diluted serially with a sterile 0.85 % (w/v) NaCl solution, and these dilutions were plated onto 0.3×marine broth 2216 (MB, BD) agar plates (containing 11.22 g marine broth 2216, 15 g agar and 1 l deionized water). Single colonies were isolated after incubation at 28 Ǫ C for 1 week, and a light pink coloured colony was obtained and designated strain XAAS-2. Unless otherwise stated, strain XAAS-2 T was routinely cultured on 0.3×MB agar plates at 28 Ǫ C and preserved as a glycerol suspension (20 % v/v) at ‒80 Ǫ C. On the basis of 16S rRNA gene sequence similarities and analysis of phylogenetic trees, Pontibacter xinjiangensis CCTCC AB 207200 T, Pontibacter odishensis KCTC 23982 T, Pontibacter locisalis CCTCC AB 2015060 T and P. actiniarum KACC 15405 T were used as reference strains for phenotypic characterization and fatty acid analyses. P. xinjiangensis CCTCC AB 207200 T was also used as a reference strain for polar lipid analyses and DNA– DNA hybridization.Author affiliations: 1 Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, PR China; 2 Xingjian Laboratory of Special Environmental Microbiology, Urumqi 830091, PR China.*Correspondence: Kai Lou, loukai02@mail.tsinghua.edu.cnKeywords: Pontibacter; rhizosphere soil; Tamarix.Abbreviations: APL, aminophospholipid; GL, glycolipid; MK-7, menaquinone 7; PE, phosphatidylethanolamine; PL, phospholipid; UL, unidentified lipid. †These authors contributed equally to this work.The GenBank accession number for the 16S rRNA gene sequence of strain XAAS-2 T is MF410355.Two supplementary figures are available with the online version of this article.Cell morphology was examined by phase-contrast microscopy (Olympus CX41) and by transmission electron microscopy (Hitachi H-600A) after incubation in 0.3×MB at 28 Ǫ C for 2 days by shaking at 150 r.p.m. Gram staining was carried out using the standard Gram reaction and confirmed by using the KOH lysis test method [11]. Catalase and oxidase activities were tested by bubble formation in 3.0 % (v/v) H 2 O 2 solution and a bioMerieux oxidase reagent kit, respectively. Gliding motility was investigated as described by Bowman [12]. The presence of flexirubintype pigments was detected as described previously [13]. Growth at 4, 5, 6, 10, 15, 20, 28, 30, 35, 37, 40 and 45 Ǫ C was measured by observing the formation of colonies on 0.3×MB agar plates for up to 2 weeks to estimate the optimal temperature and temperature range for growth. Salt tolerance was examined at different NaCl concentrations (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % w/v) in the basic medium of R2A broth (0.25 g tryptone, 0.5 g acid-hydrolysed casein, 0.5 g yeast extract powder, 0.5 g soluble starch, 0.3 g K 2 HPO 4, 0.0 g MgSO 4, 0.3 g sodium pyruvate, 0.25 g peptone, 0.5 g glucose, 1 l distilled water, pH 7.2±0.2) and at different NaCl concentrations (0.6, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % w/v) in 0.3×MB at 28 Ǫ C. The pH range (between pH 4.5 and 10.5, with 0.5 pH unit increments) and optimum pH for growth were determined in R2A broth supplemented with 1 % NaCl (w/v) and 0.3×MB by using the buffer systems and methods described previously [9, 14]. Anaerobic growth was determined as described previously [9]. Antibiotic sensitivity was investigated on 0.3×MB agar plates by using discs (Binhe) containing different antibiotics for 5 days at 28 Ǫ C. Nitrate reduction was determined as described by Lanyí [15]. Hydrolysis of starch, casein and gelatin was investigated according to previously described methods [16]. Other biochemical tests were carried out using API (20NE, 20E, API 50 CH and ZYM) systems (bio-Merieux). Oxidization of various substrates was determined by using Biolog GNIII MicroPlate assays as recommended by the manufacturer.Cells of strain XAAS-2 T were Gram-stain-negative, aerobic, oval-shaped (0.3–0.4 µm wide and 0.6 µm long), without flagella and non-motile (Fig. S1, available in the online version of this article). Colonies were light pink, circular, shiny, convex, smooth and 1–2 mm in diameter after 5 days of incubation at 28 Ǫ C on 0.3×MB agar. Strain XAAS-2 T was positive for oxidase and catalase activities. Cells grew at 5–37 Ǫ C (optimum, 28–30 Ǫ C), pH 6.0–9.5 (optimum, pH 7.0–8.0) and 0–8 % (w/v) NaCl (optimum, 0.5–2.0 %). Flexirubin-type pigments were not detected. Strain XAAS-2 T was susceptible to tetracycline, streptomycin, ampicillin, penicillin, ceftriaxone, lincomycin, TMP oxygen pyrimidine, rifampicin, clindamycin, neomycin, chloramphenicol, erythromycin, norfloxacin, vancomycin, acetylspiramycin, cefotaxime and ofloxacin, but resistant to gentamicin, kanamycin and tobramycin. Detailed phenotypic characteristics are given in Table 1 and in the species description.For 16S rRNA gene sequencing, phylogenetic analysis and G+C content determination, genomic DNA was extracted from fresh culture of strain XAAS-2 T using a ZR Fungal/ Bacterial DNA Kit (Zymo Research). PCR amplification of the 16S rRNA gene was carried out using two universal primers: 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGAC-3′) [17]. The purified PCR product was ligated into pEASY -T1 vector (TransGen Biotech), cloned and sequenced according to the manufacturer’ s instructions. We thus obtained nearly fulllength 16S rRNA gene sequences of 1489 bp. The determined 16S rRNA gene sequence was submitted to the GenBank database. Preliminary screening for similarity was done by using the BLAST algorithm, and it was also confirmed by accessing EzTaxon-e BLAST analysis [18].The nucleotide sequence of XAAS-2 T displayed 93.9– 97.1 % identity to those of other species of the genus Pontibacter. The most closely related strain was P. xinjiangensis CCTCC AB 207200 T, and all other validly published species expressed 16S rRNA gene sequence similarities lower than 97.0 %. The phylogenetic trees were reconstructed from the alignment the of XAAS-2 T 16S rRNA gene sequence and those of closely related species of the genus Pontibacter by using the neighbour-joining, maximum-likelihood and maximum-evolution algorithms implemented in MEGA software, version 7 [19]. The topologies of the phylogenetic trees were evaluated using the bootstrap resampling method with 1000 replicates. The G+C content of genomic DNA was determined using the high performance liquid chromatography (HPLC) method [20]. We found that the 16S rRNA gene of strain XAAS-2 T falls into a distinct evolutionary branch on the phylogenetic trees (Fig. 1). This indicates that the novel bacterium likely represents a novel species belonging to the genus Pontibacter. The DNA G+C content of strain XAAS-2 T was 50.6 mol%.DNA–DNA hybridization is considered as a benchmark to define bacterial species [21]. In this study, to evaluate DNA–DNA relatedness between strain XAAS-2 T and its closest phylogenetic neighbour, P. xinjiangensis CCTCC AB 207200 T, DNA–DNA hybridization experiments were carried out by the optical renaturation rate method [22] using a Perkin Elmer Lambda 35 UV/VIS spectrophotometer at the China Centre for Industrial Culture Collection (Beijing, China). Analysis of DNA–DNA hybrids performed in triplicate showed that isolate XAAS-2 T exhibited mean DNA– DNA relatedness of 47.5 % (SD=3.27) to the type strain P. xinjiangensis CCTCC AB 207200 T, clearly below the 70 % threshold value generally accepted for species delineation [23], indicating that strain XAAS-2 T represents a novel species of the genus Pontibacter.Isoprenoid quinones and polar lipids were obtained from cells grown in 0.3×MB agar for 2 days at 28 Ǫ C. The quinones were extracted by using the method of Collins [24] and separated by HPLC [25]. Polar lipids were extracted from 100 mg of freeze-dried cells using the method described by Minnikin et al. [26] and analysed by thin-layer chromatography (TLC) by using chloroform/methanol/ water (65: 25: 4 by volume) in the first direction and chloroform/acetic acid/methanol/water (80: 18: 12: 5 by volume) in the second direction. Polar lipids were visualized by spraying the TLC plate with 5 % molybdophosphoric acid. Ninhydrin, Dragendorff’ s reagent, Oi -naphthol-sulfuric acid and molybdenum blue reagent were used as specific detection reagents.Table 1. Phenotypic comparison of strain XAAS-2T and reference strains Strains/species: 1, Pontibacter brevis XAAS-2T; 2, Pontibacter xinjiangensis CCTCC AB 207200T; 3, Pontibacter odishensis KCTC 23982T; 4, Pontibacter locisalis CCTCC AB 2015060T; 5, Pontibacter actiniarum KACC 15405T. All strains are positive for catalase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N -acetyl- β -glucosaminidase and hydrolysis of aesculin and gelatin. All strains are negative for indole production, glucose acidification, arginine dihydrolase, urease, H2S production, Voges–Proskauer test, lysine decarboxylase, ornithine decarboxylase, lipase (C14), Oi -mannosidase, β -Fucosidase and assimilation of D- glucose, L- arabinose, D- mannose, D- mannitol, N -acetyl-glucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. All strains were cultured on 0.3 MB agars at 28 Ǫ C. Data for reference strains were obtained in this study × unless otherwise indicated. +, Positive; ‒, negative.Characteristic 1 2 3 4 5 Temperature range (Ǫ C) 5–37 4–37 10–40* 15–40† 6–43‡ Salinity (NaCl concentration, %) 0–8 0–5 0–9* 0.5–5† 0–10‡ Motility ‒ + + ‒ + Oxidase + ‒ + ‒ + Nitrate reduction ‒ + ‒ ‒ ‒ Hydrolysis of: Starch + + + + ‒ Casein ‒ + + ‒ ‒ API ZYM (enzymes): Trypsin + + + ‒ + Oi -Chymotrypsin + + ‒ ‒ ‒ Oi -Galactosidase + + ‒ ‒ ‒ β -Galactosidase + + ‒ ‒ ‒ β -Glucuronidase + ‒ ‒ ‒ ‒ Oi -Glucosidase ‒ + + ‒ + β -Glucosidase ‒ + + ‒ ‒ DNA G+C content (mol%) 50.6 47.8§ 47.5* 47.7† 48.7‡*Data taken from Subhash et al. [2]. †Data taken from Zhou et al. [3]. ‡Data taken from Nedashkovskaya et al. [1]. §Data taken from Wang et al. [4].Chemotaxonomic data supported the result of the phylogenetic analysis. The sole respiratory quinone found in strain XAAS-2 T was MK-7, in line with observations in all Pontibacter species. The major polar lipids were PE and two unidentified lipids (UL 1 and UL 3). Analysis of the available data revealed the polar lipids of different species in the genus of Pontibacter are highly divergent, but all species with validly published names possess PE as one of the major polar lipids [27]. In addition, minor amounts of three aminophospholipids (APL 1, APL 2, and APL 3), one unidentified glycolipid (GL), one unidentified phospholipid (PL) and three unidentified lipids (UL 2, UL 4, and UL 5) were also present in strain XAAS-2 T. The polar lipids of strain XAAS-2 T were similar to those of the type strain of P. xinjiangensis CCTCC AB 207200 T in that PE and two unidentified lipids, UL 4 and UL 6, were the major polar lipids (Fig. S2).Cellular fatty acids methyl esters were obtained from cells grown in 0.3×MB agar for 2 days at 28 Ǫ C and analysed by using gas chromatography (6850, Agilent) and the TSBA6 database according to the instructions of the Microbial Identification System (MIDI). The major fatty acids found in strain XAAS-2 T were summed feature 4 (iso-C 17: 1 I and/or anteiso-C 17: 1 B) and iso-C 15: 0. The fatty acid profile of strain XAAS-2 T was similar to those of the type strains of three phylogenetically closely related Pontibacter species and P. actiniarum, although there were differences in the proportions of some fatty acids (Table 2).Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that XAAS-2 T represented a member of the genus Pontibacter, with sequence similarities (&lt;97.1 %) lower than the suggested value (98.7 %) for species demarcation [28]. A number of phenotypic characteristics also supported the distinction of XAAS-2 T from its closest relative P. xinjiangensis CCTCC AB 207200 T, namely, the inability to hydrolyse casein, the inability to reduce nitrates and activity levels of several enzymes. Analysis of DNA–DNA hybrids performed in triplicate showed that isolate XAAS-2 T exhibited mean DNA–DNA relatedness of 47.5 % (SD=3.27) to the most closely related type strain P. xinjiangensis CCTCC AB 207200 T, clearly below the 70 % threshold value generally accepted for species delineation [23], further confirming that XAAS-2 T represents a novel genomic species. Conclusively, the data from this study indicate that strain XAAS-2 T represents a novel species of the genus Pontibacter. The name Pontibacter brevis sp. nov. is proposed for this isolate, with XAAS-2 T as its type strain.