Phytoactinopolyspora alkaliphila sp. nov., an alkaliphilic actinomycete ...

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2058–2063

DOI 10.1099/ijsem.0.000992

Phytoactinopolyspora alkaliphila sp. nov., an alkaliphilic actinomycete isolated from a saline-alkaline soil Yong-Guang Zhang,1 Xin-Hua Lu,2 Yan-Bo Ding,2 Xing-Kui Zhou,3 Li Li,1 Jian-Wei Guo,1,4 Hong-Fei Wang,1,5 Yan-Qing Duan3 and Wen-Jun Li1,6 Correspondence Wen-Jun Li

1

[email protected] or

2

[email protected]

Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, U¨ru¨mqi 830011, PR China New Drug Research & Development Center, North China Pharmaceutical Group Corporation, National Microbial Medicine Engineering & Research Center, Shijiazhuang 050015, PR China

3

China Tobacco Yunnan Industrial Co, Ltd, Kunming 650231, PR China

4

Key Laboratory of Higher Quality and Efficient Cultivation and Security Control of Crops for Yunnan Province, Honghe University, Mengzi 661100, PR China

5

College of Life Science, Liaoning Normal University, Dalian 116029, PR China

6

State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China

An alkaliphilic, filamentous actinomycete, designated EGI 80629T, was isolated from a soil sample of Xinjiang, north-west China. Strain EGI 80629T grew at pH 6.0–11.0 (optimum pH 9.0–10.0) and in the presence of 0–13.0 % NaCl (optimum 3.0–5.0 %). The isolate formed fragmented substrate mycelia, and aerial hyphae with short spore chains with rod-like spores. Whole-cell hydrolysates of the isolate contained LL -diaminopimelic acid as the diagnostic diamino acid, and mannose and rhamnose as diagnostic sugars. The major fatty acids identified were iso-C15 : 0, iso-C16 : 0, anteiso-C15 : 0 and iso-C17 : 0. The predominant menaquinone was MK-9(H4), while the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two phosphatidylinositol mannosides, five unknown phospholipids, three unknown phosphoglycolipids, one unknown glycolipid, four unknown polar lipids and one unknown aminophospholipid. The G+C content of the genomic DNA was 67.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain EGI 80629T clustered with the genus Phytoactinopolyspora. The 16S rRNA gene sequence similarity between strain EGI 80629T and Phytoactinopolyspora endophytica EGI 60009T was 96.8 %. Based on morphological, chemotaxonomic and phylogenetic characteristics, strain EGI 80629T represents a novel species of the genus Phytoactinopolyspora, for which the name Phytoactinopolyspora alkaliphila sp. nov. is proposed. The type strain is EGI 80629T (5CGMCC 4.7225T5KCTC 39701T).

The genus Phytoactinopolyspora was newly proposed as a member of the family Jiangellaceae by Li et al. (2015). The genus differs from the genera Jiangella and Haloactinopolyspora by the formation of short spore chains and no pseudosporangium-like, rhiziform spore aggregate at Abbreviation: ISP, International Streptomyces Project. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain EGI 80629T is KU042078. Two supplementary tables are a supplementary figure are available with the online Supplementary Material.

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maturity, and by specific 16S rRNA gene signature nucleotides (Li et al., 2015). At the time of writing, the genus Phytoactinopolyspora consists of only one species with a validly published name, Phytoactinopolyspora endophytica. It is very interesting that members of the family Jiangellaceae are distributed in several environments, including medicinal plants (Qin et al., 2009; Li et al., 2015), an indoor environment (Ka¨mpfer et al., 2011), a cave (Lee, 2008), a salt lake (Tang et al., 2011), desert soil (Song et al., 2005) and saline-alkaline soil (Zhang et al., 2014), despite there only being seven species in the family with validly published names (http://www.bacterio.net/).

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Printed in Great Britain

Phytoactinopolyspora alkaliphila sp. nov.

Simultaneously, these species show different preferences for alkalinity or salts. Haloactinopolyspora alba is halophilic (Tang et al., 2011), Jiangella alkaliphila (Lee, 2008) and Haloactinopolyspora alkaliphila (Zhang et al., 2014) are alkaliphilic, and other species like normal cultural conditions. During a program on diversity of alkaliphilic actinobacteria, a novel alkaliphilic strain, designated EGI 80629T, was isolated from a saline-alkaline soil of Xinjiang, north-west China. In this study, strain EGI 80629T was characterized by a polyphasic approach, and the obtained data suggest that the isolate represents a novel species of the genus Phytoactinopolyspora. Strain EGI 80629T was obtained from a desert soil sample (collected from Karamay, Xinjiang, north-west China) after incubation for 3 weeks at 30 8C on International Streptomyces Project (ISP) 2 medium (Shirling & Gottlieb, 1966) modified by adjusting the pH to 10.0 with autoclaved 10 M NaOH. The same medium was also used for cultivating and maintaining the purified isolate. Strain EGI 80629T was preserved as glycerol suspensions (20 %, w/v) at 280 8C. For morphological observations, strain EGI 80629T was cultured for 14–28 days at 30 8C on ISP 2 modified with addition of 3.0 % (w/v) NaCl and adjusting the pH to 10.0 with 10 M NaOH. Morphological features were observed by light microscopy (BH-2; Olympus) and scanning electron microscopy (Quanta 200; FEI). Cultural features were determined after incubation for 2 and 4 weeks at 30 8C according to the methods of Shirling & Gottlieb (1966). The colours of the aerial and substrate mycelia were determined with the ISCC–NBS colour charts (Kelly, 1964). Carbon-source utilization tests were performed according to the methods described by Shirling & Gottlieb (1966). Nitrogen-source utilization tests were analysed as described by Williams et al. (1983). All media were adjusted to pH 10.0 with 10 M NaOH.

(a)

NaCl tolerance tests were performed on R2A agar modified by the addition of various concentrations of NaCl (0–15 %, at intervals of 1 %, w/v) and adjusting pH to 10.0 with 10 M NaOH. The temperature range for growth was examined at 5–60 8C (at intervals of 5 8C) on R2A with the addition of 3.0 % (w/v) NaCl and adjusting pH to 10.0 with 10 M NaOH; cultures were incubated for 14–28 days at 30 8C. Growth at different pH (pH 4.0– 12.0, at intervals of 1.0 pH unit) was examined in R2A broth supplemented with 3.0 % NaCl (w/v) using the buffer system described by Xu et al. (2005). Physiological and biochemical characteristics were examined as described previously (Goodfellow, 1971; Williams et al., 1983). Strain EGI 80629T formed well-differentiated substrate mycelium, which fragmented into short or long rods at the early growth stage. The isolate also produced white aerial hyphae on yeast extract-malt extract agar (ISP 2), potato-dextrose-agar (PDA) and nutrient agar; the hyphae fragmented into rod-like-spore chains, and the spore chains did not aggregate at maturity (Fig. 1). Strain EGI 80629T grew well on ISP 2 and PDA, moderately on nutrient agar and oatmeal agar (ISP 3), and weakly on inorganic starch agar (ISP 4), glycerol-asparagine agar (ISP 5), Czapek’s agar and Gauze No. 1 agar. No soluble pigments were observed on any of the media tested. The cultural characteristics of the strain are depicted in Table S1 (available in the online Supplementary Material). Strain EGI 80629T could grow at pH 6.0–11.0 and 15–45 8C, with optimum growth at pH 9.0–10.0 and 30 8C. The isolate could tolerate up to 13.0 % (w/v) NaCl and grow optimally in the presence of 3.0–5.0 % (w/v) NaCl. These results indicated that strain EGI 80629T is moderately alkaliphilic and halotolerant. The reactions for catalase and gelatin liquefaction were positive, but negative for oxidase activity, nitrate reduction, coagulation and peptonization of skimmed milk and production of

(b)

Fig. 1. Scanning electron micrograph of spore chains of strain EGI 80629T grown for 4 weeks at 30 8C on ISP 2 agar modified with 3 % (w/v) NaCl added and pH adjusted to 10.0 with 10 M NaOH. (a), Fragmented substrate mycelium; (b), aerial hyphae with rod-like spore chains. Bars, 5 mm. http://ijs.microbiologyresearch.org

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T 95 Jiangella muralis 15-Je-017 (FN645214) T *52 Jiangella alba YIM 61503 (FJ157186) 0.01 *99 Jiangella alkaliphila D8-87T (AM422451) *79 Jiangella gansuensis YIM 002T (AY631071) Haloactinopolyspora alba YIM 93246T (FJ969846) *100 Haloactinopolyspora alkaliphila EGI 80088T (KF040422) *100 Phytoactinopolyspora endophytica EGI 60009T (KP271925) 71 Phytoactinopolyspora alkaliphila EGI 80629T (KU042078) *98 Actinopolymorpha rutila YIM 45725T (EF601829) *100 Actinopolymorpha singaporensis IM 7744T (AF237815) Kribbella koreensis IMSNU 50530T (Y09159) *56 *60 Kribbella antibiotica YIM 31530T (AY082063) *100 Kribbella sandramycini ATCC 39419T (AF005020) Kribbella solani DSA1T (AY253862) *80 Kribbella jejuensis HD9T (AY253866) Aeromicrobium alkaliterrae KSL-107T (AY822044) *99 *100 Aeromicrobium fastidiosum KCTC 9576T (AF005022) Aeromicrobium tamlense SSW1-57T (DQ411541) Nocardioides plantarum NCIMB 12834T (AF005008) *71 Nocardioides luteus KCTC 9575T (AF005007) *100 *79 Nocardioides albus KCTC 9186T (AF004988) Nocardioides nitrophenolicus NSP41T (AF005024) *76 Nocardioides aromaticivorans H-1T (AB087721) *100 Nocardioides simplex KCTC 9106T (AF005009) *65 Streptomyces sparsogenes NRRL 2940T (AJ391817)

Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain EGI 80629T. Bootstrap values (expressed as percentages of 1000 replications) .50 % are shown at the branch nodes. Bar, 0.01 sequence divergence. Asterisks indicate that the clades were conserved when maximum-parsimony and maximum-likelihood methods were used to construct the phylogenetic trees.

H2S. Strain EGI 80629T could hydrolyse Tweens 20, 40, 60 and 80, but not cellulose or starch. Biomass for chemical and molecular studies was obtained by cultivation at 30 8C for 6 days in shake flasks (about 150 r.p.m.) containing ISP 2 broth adjusted to pH 10.0 with NaOH. Diaminopimelic acid isomers of whole-cell hydrolysates were analysed by TLC as described previously (Staneck & Roberts, 1974). Whole-cell sugars were detected by HPLC after pre-column derivatization with 1-phenyl-3-methyl-5pyrazolone (PMP) (Tang et al., 2009). For identification of the mycolic acids present in the cell wall, one-dimensional TLC was carried out following the standard procedure described by Minnikin et al. (1975). Polar lipids were extracted by two-dimensional TLC and identified following the method of Minnikin et al. (1984). Menaquinones were extracted and purified according to Collins et al. (1977) and analysed by HPLC (Groth et al., 1996). For fatty acid analyses, strain EGI 80629T was cultured for 4 days at 30 8C on TSB medium modified by the addition of 0.5 % (w/v) NaCl and adjusting pH to 10.0 with NaOH. Cellular fatty acids analysis was performed as described by Sasser (1990) according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System. For determination of G+C content, the genomic DNA of strain EGI 80629T was prepared according to Marmur (1961), and the G+C content was determined by the HPLC method (Mesbah et al., 1989). 2060

Strain EGI 80629T contained LL -diaminopimelic acid as diagnostic cell-wall diamino acid. Whole-cell hydrolysates contained mannose and rhamnose as the diagnostic sugars, as well as three unknown sugars as minor sugars. Mycolic acids were absent in the cell wall of strain EGI 80629T. The predominant menaquinone detected was MK-9(H4). The polar lipids were diphosphatidylglycerol, two phosphatidylinositol mannosides, phosphatidylglycerol, three unknown phosphoglycolipids, one unknown glycolipid, five unknown phospholipids, four unknown polar lipids and one unknown aminophospholipid (Fig. S1). Strain EGI 80629T contained branched saturated fatty acids as major fatty acids (.70 %). The main fatty acids (.10 %) were iso-C15 : 0, iso-C16 : 0, anteiso-C15 : 0 and iso-C17 : 0, and the other fatty acids are given in Table S2. The DNA G+C content of strain EGI 80629T was 67.3 mol%. Genomic DNA preparation, PCR amplification and sequencing of the 16S rRNA gene were carried out using procedures described by Li et al. (2007). Multiple alignments with sequences of the members of the order Jiangellales, and calculations of levels of sequence similarity were carried out using the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al., 2012) on the basis of 16S rRNA gene sequence data. Phylogenetic analysis was performed using three tree-making algorithms, neighbour-joining (Saitou & Nei, 1987), maximum-

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likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971), using the software MEGA 6 (Tamura et al., 2013). The topologies of the resultant trees were evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. The almost-complete 16S rRNA gene sequence (1518 bp) was determined for strain EGI 80629T. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the strain fell within the members of the order Jiangellales. The 16S rRNA gene sequence similarities between strain EGI 80629T and Phytoactinopolyspora endophytica EGI 60009T, Jiangella alba YIM 61503T, Jiangella muralis 15-Je-017T, Jiangella gansuensis YIM 002T, Jiangella alkaliphila D8-87T, Haloactinopolyspora alkaliphila EGI 80088T and Haloactinopolyspora alba YIM 93246T were 96.8 %, 96.6 %, 96.6 %, 96.2 %, 95.9 %, 96.2 % and 95.5 %, respectively. Strain EGI 80629T formed a distinct clade with P. endophytica EGI 60009T with a high bootstrap value (98 %), while other members of the order Jiangellales formed another clade (Fig. 2). This relationship was also supported by the other two tree-making methods used in this study (data not shown). In addition to the close phylogenetic relationship with P. endophytica EGI 60009T, strain EGI 80629T has some typical characteristics of the genus Phytoactinopolyspora including well-differentiated aerial hyphae and spore chains not aggregating at maturity, diagnostic cell-wall amino acid (LL -diaminopimelic acid), absence of mycolic acids, major fatty acids (iso-C15 : 0, anteiso-C15 : 0 and isoC17 : 0), the main phospholipids (diphosphatidylglycerol and phosphatidylinositol mannosides) and MK-9(H4) as the predominant menaquinone (Li et al., 2015). However, strain EGI 80629T differs greatly from P. endophytica EGI 60009T by its alkaliphilic growth, biochemical and physiological features and drug resistance as shown in Table 1, chemotaxonomic characteristics including fatty acid profile (Table S1), cell-wall sugars and DNA G+C content. Strain EGI 80629T contains mannose and rhamnose as diagnostic sugars, while those for P. endophytica EGI 60009T were mannose, rhamnose, glucose and galactose, which is coincident with the previous reports (Li et al., 2015). Furthermore, the genomic DNA G+C content of strain EGI 80629T is 67.3 mol%, which is lower than that of the strain P. endophytica EGI 60009T (Li et al., 2015). In conclusion, strain EGI 80629T is clearly different from its closest phylogenetic neighbour P. endophytica EGI 60009T by phenotypic, chemotaxonomic and phylogenetic data, and the isolate therefore represents a novel species of the genus Phytoactinopolyspora, for which the name Phytoactinopolyspora alkaliphila sp. nov. is proposed.

Table 1. Phenotypic characteristics that distinguish strain EGI 80629T from its closest phylogenetic neighbour P. endophytica EGI 60009T Strains: 1, EGI 80629T; 2, P. endophytica EGI 60009T. Both strains can use D -fructose, D -galactose, D -glucose, D -inositol, lactose, DL -malic acid, maltose, D -mannose, L -rhamnose, sucrose, trehalose, D -xylose and sodium citrate as sole carbon sources, but not D -ribose. DL -Alanine, L -asparagine, L -aspartic acid, L -cystine, L -glutamic acid, L -histidine, L -isoleucine, L -lysine, L -methionine, L -phenylalanine, L -serine, L -threonine and L -valine can be used as sole nitrogen sources for growth, but not L -arginine. Both strains show positive reactions for catalase and hydrolysis of Tween 80, but negative reactions for H2S production, hydrolysis of cellulose and starch, and coagulation and peptonization of skimmed milk. Both strains are sensitive to ampicillin (10 mg), amphozone (30 mg), chloramphenicol (30 mg), gentamicin (10 mg), lincomycin (2 mg), novobiocin (30 mg), penicillin G (10 mg) and polymyxin B (300 mg), and resistant to azithromycin (15 mg), clarithromycin(15 mg), erythromycin (15 mg), kanamycin (30 mg), neomycin (30 mg) and roxithromycin (15 mg).+, Positive/ utilized; 2, negative/not utilized. Characteristic Growth conditions pH range (optimum) NaCl range (optimum) (%, w/v) Temperature range (optimum) Physiological features Nitrate reduction Oxidase Gelatin liquefaction Hydrolysis of Tweens 20, 40 and 60 Carbon source utilization D -Arabinose Glycerol Raffinose D -Sorbitol L -Sorbose D -Xylitol Sodium pyruvate Nitrogen source utilization L -Tryptophan L -Tyrosine Drug resistances Cefalotin (30 mg) Spectinomycin(100 mg) Streptomycin(10 mg) Tetracycline (30 mg) Vancomycin (30 mg)

1

2

6–11 (9–10) 0–13 (3–5) 15–45 (30)

5–9 (7–8) 0–10 (2–3) 15–35 (30)

2 2 + +

+ + 2 2

+ + + + + + +

2 2 2 2 2 2 2

+ 2

2 +

2 2 2 + 2

+ + + 2 +

Description of Phytoactinopolyspora alkaliphila sp. nov.

alkali alkali; Gr. adj. philos loving; N.L. fem. adj. alkaliphila loving alkaline conditions).

Phytoactinopolyspora alkaliphila (al.ka.li9phi.la. Arabic article al the; Arabic n. qaliy ashes of saltwort; N.L. n.

Gram-stain-positive, aerobic actinomycete. Well-developed substrate mycelium which exhibits fragmentation into

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short or elongated rods at early stage of growth; fragmented aerial mycelium with short chains of spores, and spore chains do not aggregate at maturity. Grows well on ISP 2 and PDA, moderately on nutrient agar and ISP 3, and weakly on ISP 4, ISP 5, Cazpek’s agar and Gauze No. 1 modified with pH adjusted to 10.0 with autoclaved 10 M NaOH. Diffusible pigment is not observed on any of the media tested. Colours of aerial mycelium and substrate mycelium on ISP2 agar medium are while and pale yellow, respectively. Growth occurs at pH 6.0–11.0, with 0–13.0% (w/v) NaCl and at 15–45 uC; optimum growth occurs at pH 9.0–10.0, with 3.0–5.0% NaCl and at 30 uC. D -Arabinose, D -fructose, D -galactose, D -glucose, glycerol, D -inositol, lactose, DL -malic acid, maltose, D -mannose, raffinose, L -rhamnose, D -sorbitol, L -sorbose, sucrose, trehalose, D -xylose, D -xylitol, sodium citrate and sodium pyruvate can be utilized as sole carbon sources for growth, but not D -ribose. DL -Alanine, L -asparagine, L -aspartic acid, L -cystine, L -glutamic acid, L -histidine, L -isoleucine, L -lysine, L -methionine, L -phenylalanine, L -serine, L -threonine, L -tryptophan and L -valine can be used as sole nitrogen sources for growth, but not L -arginine or L -tyrosine. Positive reactions for catalase and gelatin liquefaction, but negative reactions for oxidase, coagulation and peptonization of skimmed milk, nitrate reduction and H2S production. Hydrolyses Tweens 20, 40, 60 and 80, but not cellulose or starch. Whole-cell hydrolysates contain LL diaminopimelic acid as the diagnostic cell-wall amino acid, and mannose and rhamnose as diagnostic sugars. Mycolic acids are absent. The predominant menaquinone detected is MK-9(H4). The polar lipids are diphosphatidylglycerol, two phosphatidylinositol mannosides, phosphatidylglycerol, three unknown phosphoglycolipids, one unknown glycolipid, five unknown phospholipids, four unknown polar lipids and one unknown aminophospholipid. The major fatty acids are iso-C15:0, iso-C16:0, anteiso-C15:0 and iso-C17:0. The type strain is EGI 80629T (5CGMCC 4.7225T5KCTC 39701T) isolated from a soil sample collected in Karamay, Xinjiang, north-west China. The G+C content of the DNA of the type strain is 67.3 mol%.

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Acknowledgements This research was supported by the joint fund of the National Natural Science Foundation of China (NSFC) (nos U1403101 and 31400009), the West Light Foundation of Chinese Academy of Sciences (RCPY201203) and Xinjiang Uygur Autonomous Region Natural Science Foundation (2014211A074). W.-J. L. was also supported by the ‘Hundred Talents Program’ of the Chinese Academy of Sciences, the High-level Talents Program of Xinjiang Autonomous Region and Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014).

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