Altererythrobacter Rhizovicinus Sp. Nov., Isolated from Rhizosphere Soil of Haloxylon Ammodendron

TAXONOMIC DESCRIPTION Li et al., Int. J. Syst. Evol. Microbiol. 2020;70:680–686 DOI 10.1099/ijsem.0.003817

Altererythrobacter rhizovicinus sp. nov., isolated from rhizosphere soil of Haloxylon ammodendron

Hui-Ping Li, Dan Yao, Kun-Zhong Shao, Qing-Qing Han, Jing-Yi Gou, Qi Zhao* and Jin-Lin Zhang*

Abstract A salt-tolerant, Gram-negative, rod-shaped and yellow-pigmented bacterium, designated strain AY-3R T, was isolated from rhizosphere soil of a desert xerophyte, Haloxylon ammodendron, sampled at Badain Jaran Desert, Alxa region, Inner Mongolia, PR China. Growth of this strain was observed at 20–42 °C (optimum, 28–30 °C), at pH 6.0–9.0 (optimum, pH 6.0–7.0) and at 0–8 % (w/v) NaCl (optimum, 3 %). Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain AY-3RT was a member of the genus Altererythrobacter, with the highest similarity to Altererythrobacter aerophilus Ery1T (97.6 %), followed T by Altererythrobacter xinjiangensis S3-63 (96.9 %). The predominant fatty acids (>10.0 %) were C18 : 1ω7c, C17 : 1ω6c and summed

feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid and one unknown polar lipid. The predominant respiratory quinone was ubiquinone-10. The G+C content of the genomic DNA of strain AY-3R T was 66.3 mol%. On the basis of the data from this polyphasic taxonomic study, strain AY-3R T represents a novel species of the genus Altererythrobacter, named Altererythrobacter rhizovicinus sp. nov. (=MCCC 1K03572T=KCTC 72280T).

The family Erythrobacteraceae belonged to the order of Sphin- and ubiquinone-10, respectively [6]. The predominant polar gomonadales, the class of Alphaproteobacteria in the phylum lipids are diphosphatidylglycerol (DPG), phosphatidyletha- of Proteobacteria [1, 2]. At the time of writing, the family nolamine (PE), phosphatidylglycerol (PG) and sphingogly- Erythrobacteraceae contained genera with validly published colipid (SGL) [20]. The DNA G+C content is in the range of names, and the genus Altererythrobacter is one of them [2]. 52.0–69.0 mol%. The aim of this study was to describe a novel Subsequently, characteristics of the genus Altererythrobacter strain in the genus Altererythrobacter based on a polyphasic were revised by Xue et al. [3, 4]. The type species of this genus approach. is Altererythrobacter epoxidivorans, isolated from cold-seep T sediment [2]. Until now, 42 species have been identified in Strain AY-3R was isolated from the rhizosphere of a desert the genus Altererythrobacter (www.bacterio.net/-allnamesac. xerophyte, Haloxylon ammodendron, collected at Badain html). Most of them were isolated from marine environments Jaran Desert, Alxa region, Inner Mongolia, PR China (39° 23′ [2, 5–14]. The remaining species were isolated from terrestrial 7″ N, 102° 47′ 40″ E). The soil sample was serially diluted with environments, such as desert sand [4, 15, 16], permafrost sterile 0.9 % NaCl (w/v) solution, then dilutions were spread [17], mountain soil [18], rhizosphere soil [19] and forest soil on Reasoner’s 2A (R2A) agar (Difco) and incubated at 25 °C [20]. Most cells of members of the genus Altererythrobacter aerobically for 7 days. Single colonies were picked and further are Gram-reaction- negative, aerobic, motile or non-motile purified. Finally, the purified isolate was preserved as a glyc- and generally rod-shaped cells, which form yellow colonies. erol suspension (20 %, v/v) at −80 °C. Two closest type strains, T The dominant fatty acid and respiratory quinone are 18C : 1ω7c Altererythrobacer aerophilus Ery1 and Altererythrobacter

Author affiliations: 1State Key Laboratory of Grassland Agro-ecosystems; Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs; Engineering Research Center of Grassland Industry, Ministry of Education; College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, Gansu Province, PR China. *Correspondence: Qi Zhao, qzhao@ lzu. edu. cn; Jin-Lin Zhang, jlzhang@ lzu. edu. cn Keywords: Altererythrobacter rhizovicinus sp. nov.; Phylogenetic analysis; Polyphasic taxonomic. Abbreviations: ANI, average nucleotide identity; DDH, DNA–DNA hybridization; DPG, diphosphatidylglycerol; GGDC, Genome-to- Genome Distance Calculator; GL, glycolipid; ISP 2, yeast extract–malt extract; LB, Luria–Bertani; MA, marine agar; NA, nutrient agar; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, hosphatidylglycerol; PYG, peptone–yeast–glucose; R2A, Reasoner’s 2A; SGL, sphingoglycolipid; TSA, tryptone soya agar; TY, tryptone–yeast. The GenBank/EMBL/DDBJ accession number for 16S rRNA gene sequence of strain AY-3R T is MH611372. The GenBank accession number for the whole genome sequence of strain AY-3RT is SDPV00000000. Four supplementary figures are available with the online version of this article.

xinjiangensis CCTCC AB 207166T, were obtained from Genome-to-Genome Distance Calculator 2.1 (GGDC) Xu’s lab and the China Centre for Type Culture Collection (http://ggdc.dsmz.de/distcalc2.php). (CCTCC) and used as reference strains for phenotypic tests The draft genome of strain AY-3R T was 3 007 728 bp long and and fatty acid analysis. A. aerophilus Ery1T and A. xinjian- comprised three scaffolds with an N50 value 1 680 395 bp. gensis CCTCC AB 207166T were cultured on marine agar The genome coverage was 200×. A total of 2764 proteins, 2216 (MA; Difco) and 0.3×MA (Difco) at 30 °C, respectively. three rRNAs and 46 tRNAs were predicted. The complete Phylogenetic analysis using the 16S rRNA gene sequence was 16S rRNA gene sequence was 1477 bp long and contained carried out as depicted by Zhao et al. [16]. Genomic DNA of the 16S rRNA gene sequence (1457 bp) derived from the isolate was extracted by Bacterial Genomic DNA Extrac- PCR amplification. The DNA G+C content of AY-3R T was tion kit (TianGen Biotech). The 16S rRNA gene was amplified 66.3 mol%, which fell into the range reported for members by PCR using forward primer 27F (5′-AGAGTTTGATC- of the genus Altererythrobacter (52.0–69.0 mol%). The ANI CTGGCTCAG-3′) and reverse primer 1492R (5′-GGTTAC- value between strain AY-3RT and the reference strains were CTTGTTACGACTT-3′) [21]. Purification, cloning and 88.2 and 74.0 %, respectively. These values were significantly sequencing of the 16S rRNA gene were respectively executed lower than the threshold of the species boundaries (94–96 %) using a PCR purification kit (Sangon Biotech), the pMD 19 T [32]. DDH values between strain AY-3R T and its reference Vector Cloning kit (Takara) and the Sanger method (Beijing strains, A. aerophilus Ery1T and A. xinjiangensis CCTCC AB augct dna-syn Biotechnology). Then, the almost-complete 207166T, were 34.9 and 19.8 %, respectively. The critical value 16S rRNA gene sequence was compiled with the program of genomic species identification is less than 70 % [33]. These dnaman (version 8.0, Lynnon Biosoft) [22]. The EzTaxon-e data obviously indicated that strain AY-3RT represented a server (www.ezbiocloud.net/apps; [23]) was used to calcu- novel species of the genus Altererythrobacter. late levels of sequence similarity between strain AY-3RT and Growth was assessed on R2A agar, tryptone soya agar (TSA), related strains available in GenBank (www.ncbi.nlm.nih. Luria–Bertani agar (LB agar), MA, tryptone–yeast agar (TY gov/genbank/; [24]). Evolutionary distances were calculated agar), peptone–yeast–glucose agar (PYG agar), yeast extract– using Kimura’s two-parameter model [25] and phylogenetic malt extract agar (ISP 2 agar) and nutrient agar (NA). Cell trees were reconstructed using the neighbour-joining [22], morphology was examined by transmission electron micros- maximum-likelihood [26] and maximum-parsimony [27] copy (JEM 1230, jeol) with cells from the early exponential methods with the mega 7.0 program [28]. In each case, growth phase on MA. Gram-staining was carried out on MA bootstrap analysis of 1000 replicates was performed to assess according to the manufacturer’s instructions [34]. Motility the confidence levels of the branches [29]. was examined in semi-solid medium. Growth at various On the basis of phylogenetic analysis, comparison of the temperatures (4, 10, 15, 20, 25, 28, 30, 35, 40 and 42 °C), pH 16S rRNA gene sequence of strain AY-3RT revealed the 5.0–10.0 (at intervals of 0.5 pH units) by using the following highest similarity to A. aerophilus Ery1T (97.6 %), followed biological buffer systems: 100 mM citric acid/sodium citrate T by A. xinjiangensis S3-63 (96.9 %), and other recognized (pH 5.0–6.0), 100 mM Na2HPO4/NaH2PO4 (pH 6.5–8.0), members of the genus Altererythrobacter (<96.5 %). The three 100 mM Tris/100 mM HCl (pH 8.5) and 100 mM NaHCO3/ T species living trees showed that strain AY-3R fell within the Na2CO3 (pH 9.0–10.5) [35, 36]. Tolerance to 0–9 % (w/v) NaCl cluster comprising the Altererythrobacter species (Figs 1, S1 (at intervals of 0.5 %) was determined on MA for 15 days at and S2, available in the online version of this article). Strain 28 °C. Oxidase activity was determined by 1 % (w/v) AY-3RT formed a distinct genetic lineage with A. aerophilus N,N,N′,N′,-tetramethyl-p -phenylenediamine dihydrochlo- Ery1T based on results from the neighbour-joining (Fig. 1), ride (Sigma), and catalase activity was determined by bubble maximum-likelihood methods (Fig. S1). Thus, these data production with 3 % (v/v) H2O2. Oxidation fermentation, highlighted the view that strain AY-3RT was affiliated with the Tween 80, degradation of DNA, casein, starch and cellulase genus Altererythrobacter and did not belong to any recognized were tested on MA according to standard methods [37]. species of the genus Altererythrobacter. Cellular pigments were extracted with acetone/methanol (7 : 2, v/v) from cultures grown on MA and were measured The draft genome of strain AY-3RT was determined using with a spectrophotometer in the wavelength range from paired-end reads with the Illumina HiSeq-PE150 platform 200 to 1100 nm (UV-6100S, Mapada Instruments) [12]. (Majorbio Bio-Pharm Technology). The sequencing gener- Biochemical features were evaluated using the API 20NE, ated about 1 Gb clean data. The reassembling of the reads was API ZYM and API 50CH systems (bioMérieux). GENⅢ performed using SPAdes (version 3.8.1) [30]. The full-length MicroPlates (Biolog) were used to check the utilization of 71 16S rRNA gene sequence of strain AY-3RT was annotated carbon sources, according to the instructions of the manufac- via the RNAmmer 1.2 aerver [31] from the genome. For the turer’s instructions. Cells of strain AY-3R T and the reference DNA G+C content and the average nucleotide identity (ANI) strains cultured on MA at 28 °C were used for API systems calculations, the sequence of the draft genome was upload and Biolog tests. to the ChunLab's online average nucleotide identity (ANI) calculator (www.ezbiocloud.net/tools/ani) [23]. DNA–DNA Strain AY-3RT was Gram-negative, rod-shaped (0.9–1.3×0.3– hybridization (DDH) values between strain AY-3R T and 0.5 µm; see Fig. S3). Compared with the reference strain, cells the related reference strains were calculated by using the of strain AY-3RT possessed one polar flagella and were capable

681 Li et al., Int. J. Syst. Evol. Microbiol. 2020;70:680–686

T 56 Porphyrobacter sanguineus IAM 12620 (AB021493) Erythromicrobium ramosum DSM 8510T (AF465837) 0.0100 80 Porphyrobacter tepidarius DSM 10594T (AB033328) 99 50 Porphyrobacter neustonensis DSM 9434T (AB033327) T 90 Porphyrobacter dokdonensis DSW-74 (DQ011529) Erythrobacter pelagi UST081027-248T (HQ203045) T 83 Erythrobacter citreus RE35F/1 (AF118020) 92 Erythrobacter seohaensis SW-135T (AY562219) 82 Erythrobacter aquimaris SW-110T (AY461441) 93 Erythrobacter vulgaris 022-2-10T (AY706935) Altererythrobacter maritimus HME9302T (KF385494) T 100 Altererythrobacter ishigakiensis JPCCMB0017 (AB363004) Altererythrobacter insulae BPTF-M16T (MH206217) Altererythrobacter lutipelagi GH1-16T (LT797153) Altererythrobacter luteolus SW-109T (AY739662) 92 Altererythrobacter aestiaquae HDW-31T (KJ658262) Altererythrobacter epoxidivorans JCS350T (DQ304436) Altererythrobacter marinus H32T (EU726272) 51 Altererythrobacter marensis MSW-14T (FM177586) T 87 Altererythrobacter dongtanensis JM27 (GU166344) 99 Altererythrobacter troitsensis KMM 6042T (AY676115) Altererythrobacter amylolyticus NS1T (KX601069) Qipengyuania sediminis M1T (KJ734993) 50 Altererythrobacter rigui WW3T (KP997219) 50 100 Altererythrobacter deserti THG-S3T (KY287245) T 86 Altererythrobacter aerophilus Ery1 (MG183676) Altererythrobacter rhizovicinus AY-3RT (MH611372) 66 Altererythrobacter xinjiangensis S3-63T (HM028673) 82 Altererythrobacter soli MN-1T (KT906300) Altererythrobacter spongiae HN-Y73T (MG437235) T 98 Novosphingobium endophyticum EGI 60015 (KP721487) 74 Novosphingobium pentaromativorans US6-1T (AF502400) 72 Novosphingobium marinum LA53T (KJ708552) Novosphingobium malaysiense MUSC 273T (KC907395) T 87 Croceicoccus naphthovorans PQ-2 (KF145127) Croceicoccus pelagius Ery9T (KT383844 ) 94 Croceicoccus mobilis Ery22T (KT383846) 98 Croceicoccus marinus E4A9T (EF623998) Altererythrobacter aquimixticola SSKS-13T (MK194299) Caulobacter vibrioides CB51T (AJ009957)

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain AY-3R T among closely related members of the family Erythrobacteraceae. Bootstrap values (>50 %) based on 1000 replications are shown at branch nodes. Filled circles indicate that the corresponding nodes were also formed in maximum-likelihood and maximum-parsimony trees. Caulobacter vibrioides CB51T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position. of movement by means of polar flagella. Morphological agar and MacConkey agar. It was able to grow aerobically observation indicated that colonies of strain AY-3RT on MA at 20–42 °C (optimum, 28–30 °C), at pH 6.0–9.0 (optimum, were yellow, round and smooth and about 1 mm in diameter pH 6.0–7.0) and with 0–8 % (w/v) NaCl (optimum, 3 %). after growth at 28 °C for 3 days. The methanol-soluble extracts Strain AY-3RT was positive for oxidase and catalase, but it of colonies with maximum absorption at 451 and 482 nm was negative for production of H2S and indole, hydrolysis displayed a typical spectrum of carotenoid pigment. Strain of gelatin. The detail differential phenotypic characteristics AY-3RT grew well on TSA, ISP 2 agar, NA and MA, weakly of strain AY-3RT and its closest type strains of the genus on PYG agar, R2A agar and LB agar, but did not grow on TY Altererythrobacter are given in Table 1. Strain AY-3RT was

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Table 1. Characteristics that differentiate strain AY-3RT from closely related members of the genus Altererythrobacter Strains: 1, AY-3RT; 2, Altererythrobacter aerophilus Ery1T; 3, Altererythrobacter xinjiangensis CCTCC AB 207166T. Data for all strains are from this study, except where labelled. All strains were positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, activities, assimilation of glucose, hydrolysis of aesculin. All strains were negative for lipase (C14), α-galactosidase, α-mannosidase and α-fucosidase activities, indole production, hydrolysis of DNA and assimilation of capric acid. +, Positive; −, negative; w, weakly positive.

Characteristic 1 2 3

Motility + − −

Temperature range for growth (optimum) (°C) 20–42 (28-30)) 10–45 (30-40)* 20–37 (30)†

Oxidase activity + + −

Catalase activity − + +

Hydrolysis of:

Tween 80 + − +

Cellulose − − +

Assimilation of (20NE):

Arabinose − − w

Mannose + + −

Gluconate + − −

Maltose − + +

Citrate + − −

N-Acetyl-glucosamine + w −

Adipic acid − − +

Enzyme activity (API ZYM):

Cystine arylamidase + w +

Trypsin w + w

α-Chymotrypsin + − +

Acid phosphatase − + +

β-Galactosidase − − +

α-Glucosidase w + +

β-Glucosidase − + +

β-Glucuronidase w − −

Naphthol-AS-BI-phosphohydrolase + − +

N-Acetyl-β-glucosaminidase − + −

Acid production from (API 50CH):

d-Galactose + + −

rehalose + − w

DNA G+C content (mol%) 66.3 65.4* 64.6†

*Data from Meng et al. [14]. †Data from Xue et al. [4]. distinguished from the reference type species in phenotypic The polar lipids of strain AY-3RT were extracted and sepa- characteristics, such as polar flagella, temperature, hydrolysis rated by using a chloroform/methanol system and one- and of Tween 80, enzyme activities, carbohydrate utilization and two-dimensional TLC, as described previously [38]. Merck acid production. silica gel 60 F254 aluminium-backed thin-layer plates were

683 Li et al., Int. J. Syst. Evol. Microbiol. 2020;70:680–686

Table 2. Cellular fatty acid compositions (%) of strain AY-3R T and its closest related species of the genus Altererythrobacter Strains: 1, AY-3RT; 2, Altererythrobacter aerophilus Ery1T; 3, Altererythrobacter xinjiangensis CCTCC AB 207166T. All data were obtained from this study. Values<1.0 % of the total for all strains are not shown. Therefore, percentages do not add to 100 %. nd, Not detected; tr, trace amount (<1.0 %).

Fatty acid 1 2 3

Saturated:

C14 : 0 tr tr tr

C16 : 0 6.9 6.3 4.8

C18 : 0 tr tr 1.5

Unsaturated:

C16 : 1ω5c 2.6 1.2 1.2

C17 : 1ω6c 21.4 3.5 3.3

C17 : 1ω8c 1.3 tr tr

C18 : 1ω5c 1.1 tr 1.6

C18 : 1ω7c 29.8 50.7 63.5

11-methyl C18 : 1ω7c 5.9 9.5 nd

Hydroxy:

C14 : 0 2-OH 7.2 8.9 5.4

C15 : 0 2-OH 5.7 tr tr

C16 : 0 2-OH 1.4 2.2 tr

C16 : 1 2-OH nd tr 1.4

iso-C16 : 0 3-OH nd nd 1.5

Cyclic:

C19 : 0ω8c cyclo 1.1 tr nd

Summed feature 3* 12.5 12.5 11.8

*Summed features represent two or three fatty acids that cannot be separated by GLC using the midi system. Summed feature 3 consisted of

C16 : 1ω7c and/or C16 : 1ω6c. used for TLC analysis. The plate dotted with the sample was methylation and extraction, according to standard procedures developed in the two-dimensional system, with the first [40]. Respiratory quinones were isolated and analysed from solvent system of chloroform–methanol–water (65 : 25 : 4, by lyophilized cells according to Collins’ method [41]. vol.) followed by the second solvent system of chloroform– T methanol–acetic acid–water (85 : 12 : 15 : 4, by vol.). Lipids were The major polar lipids of strain AY-3R were DPG, PC, PE, visualized after being developed in the solvent system. Total PG, SGL and one unknown polar lipid (see Fig. S4), which lipids were detected using molybdatophosphoric acid and were in accordance with reference strains, but had some specific functional groups were checked using spray reagents differences from related type strains in its unidentified lipids; T specific for defined functional groups at room temperature one reference strain (A. aerophilus Ery1 ) contained three [38], i.e. ninhydrin reagent (0.2 % solution, Sigma Life unidentified glycolipids (GL1–3) [14] and another refer- T Science), molybdenum blue spray reagent (1.3 %, Sigma Life ence strain (A. xinjiangensis S3-63 ) had seven unidentified Sciences) and α-naphthol-sulphuric acid were used to detect glycolipids (GL3–9) [14]. The dominant cellular fatty acids T aminolipids, phospholipids and glycolipids, respectively [39]. (>10.0 % of total fatty acids) of strain AY-3R were C18 : 1ω7c T Cellular fatty acids of strain AY-3R and its reference strains (29.8 %), C17 : 1ω6c (21.4 %), summed feature 3 (C16 : 1ω7c and/ T were harvested from bacterial colonies grown on MA (AY- or C16 : 1ω6c; 12.5 %). The fatty acid patterns of strain AY-3R 3RT and A. aerophilus Ery1T) and 0.3×MA (A. xinjiangensis and its reference strains are shown in Table 2. The major fatty CCTCC AB 207166T) at the logarithmic phase of growth, at acids were in accordance with those found in members of 30 °C for 3 days. The fatty acid components were analysed and the genus Altererythrobacter. Moreover, strain AY-3RT had a identified by using the Microbial Identification System (Sher- higher proportion of C17 : 1ω6c (21.4 %), which was significantly lock version 6.1; midi database, TSBA6) after saponification, higher than that in the related type strains, but the content of

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C18 : 1ω7c (29.8 %) was significantly lower than that detected in Inner Mongolia, PR China (39° 23′ 7″ N, 102° 47′ 40″ E). the reference strains. The predominant respiratory quinone of The DNA G+C content of the type strain is 66.3 mol%. The strain AY-3RT was ubiquinone-10, which was consistent with GenBank/EMBL/DDBJ accession number for 16S rRNA gene the major respiratory quinone of the genus Altererythrobacter. sequence and the whole-genome sequence of strain AY-3RT Therefore, the chemotaxonomic results for strain AY-3R T were are MH611372 and SDPV00000000, respectively. in good agreement with the characteristics of the other species belonging to the genus Altererythrobacter. Funding information T Based on the evidence presented in this study, strain AY-3R The work was financially supported by the National Natural Science should be considered to represent a novel species of the genus Foundation of China (Grant No. 31801944 and 31222053), the Natural Altererythrobacter, for which named Altererythrobacter rhizo- Science Foundation of Gansu Province, China (Grant No. 17JR5RA211), the Science and Technology Support Program of Gansu Province, China vicinus sp. nov. is proposed. (Grant No. 1604NKCA077), and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2019–41).

Acknowledgement Description of Altererythrobacter We thank Dr Xiupian Liu at the Marine Culture Collection of China and rhizovicinus sp. nov. Dr Songzhen Yang at the Guangdong Culture Collection Center of China for providing related technical support. The type strain A. aerophilus Altererythrobacter rhizovicinus (rhi.zo.vi.ci′nus. Gr. fem. n. Ery1T was a generous gift from Professor Xue-Wei Xu (Second Institute rhiza root; L. masc. adj. vicinus neighbouring; N.L. masc. adj. of Oceanography, Ministry of Natural Resources, Hangzhou 310012, rhizovicinus neighbouring a root, referring to the rhizosphere, Zhejiang Province, PR China). soil closely related to a desert plant roots, from where the type Conflicts of interest strain was isolated). The authors declare that there are no conflicts of interest. Cells are Gram-stain- negative and rod-shaped, 0.3–0.5 µm References wide and 0.9–1.3 µm long. Each cell has one polar flagella by 1. Lee KB, Liu CT, Anzai Y, Kim H, Aono T et al. 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