TAXONOMIC DESCRIPTION Ai et al., Int J Syst Evol Microbiol 2017;67:4210–4215 DOI 10.1099/ijsem.0.002284
Allobranchiibius huperziae gen. nov., sp. nov., a member of Dermacoccaceae isolated from the root of a medicinal plant Huperzia serrata (Thunb.) Meng-Jie Ai,† Ye Sun,† Hong-Min Sun, Hong-Yu Liu, Li-Yan Yu and Yu-Qin Zhang*
Abstract A Gram-stain-positive, non-spore-forming actinobacterial strain, designated CPCC 204077T, was isolated from the surfacesterilized root of a medicinal plant Huperzia serrata (Thunb.) collected from Sichuan Province, south-west China. The peptidoglycan type of strain CPCC 204077T was detected as A4a with an L-Lys–L-Ser–D-Asp interpeptide bridge. Galactose, glucose, rhamnose and ribose were the sugar compositions in the whole-cell hydrolysates. MK-8(H4) was the only menaquinone. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, one unidentified phospholipid and one unidentified glycolipid. The major fatty acid was iso-C16 : 0. The genomic DNA G+C content was 71.0 mol%. The phylogenetic tree based on 16S rRNA gene sequences showed that strain CPCC 204077T stood for a distinct lineage within the family Dermacoccaceae alongside the genera Branchiibius, Demetria and Barrientosiimonas, with the highest 16S rRNA gene sequence similarities to Branchiibius hedensis Mer 29717T (95.0 %), Calidifontibacter indicus PC IW02T (95.0 %), Barrientosiimonas humi 39T (94.9 %) and Demetria terragena HKI 0089T (94.7 %), and less than 94.7 % sequence similarities to all other species. Signature nucleotides in the 16S rRNA sequence showed that the strain contained the Dermacoccaceae family-specific 16S rRNA signature nucleotides and a genus-specific diagnostic nucleotide signature pattern. Combining the genotypic and phenotypic analyses, we propose that strain CPCC 204077T represents a novel species of a new genus in the family Dermacoccaceae with the name Allobranchiibius huperziae gen. nov., sp. nov. Strain CPCC 204077T (=NBRC 110719T=DSM 29531T) is the type strain of the type species.
The family Dermacoccaceae was first dissected by Stackebrandt and Schumann to embrace the type genus Dermacoccus as well as the genera Kytococcus and Demetria [1–3]. Recently, seven more genera, namely Luteipulveratus, Yimella, Branchiibius, Calidifontibacter, Flexivirga, Barrientosiimonas and Rudaeicoccus have been described [4–10]. Members of the family have been isolated from multiple environments, such as terrestrial habitats (Antarctic soil, soil near wastewater treatment, plant tissue), aquatic habitats (fresh water, hot springs, sea water, deep-sea sediment, branchia of Japanese codling), human blood, indoor air sample, and agar plates in the laboratory. All of them share a common taxon-specific 16S rRNA signature nucleotide pattern at positions 140–223 (C–G), 144–178 (C–G), 248– 276 (C–G), 258–268 (G–C), 379–384 (C–G), 407–435 (A– U, G–C), 502–543 (A–U), 586–755 (C–G), 602–636 (C–G), 610 (R), 612–628 (Y–G), 615–625 (G–C), 616–624 (G–Y), 630 (Y), 668–738 (A–U), 839–847 (U–A), 863 (U) and
1133–1141 (A–U) [1]. In this paper, we propose that strain CPCC 204077T represents a novel species of a new genus in the family Dermacoccaceae. The endophytic actinobacterium, designated CPCC 204077T, was isolated from the root of a medicinal plant Huperzia serrata (Thunb.) collected from low brush (29 41¢ 24.98† N, 103 10¢ 16.23† E, 1187 m H) located in Hongya County of Sichuan Province, south-west China. The plants were divided into an aerial part and an underground part which were then sterilized separately. We transferred the sterilized plant tissues into aseptic dishes on a superclean bench to dry the disinfectant attached to the surface, then cut off the ends and ground up the middle part of the plant sample in a sterilized container. The disinfectants used in this test included sodium hypochlorite (75 %, v/v) and alcohol (50 %, v/v). The filamentous plant fragments were used for isolation of endophytic strains. Tap wateryeast extract (TWYE) agar containing (l 1) (yeast extract
Author affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China. *Correspondence: Yu-Qin Zhang,
[email protected] Keywords: Allobranchiibius huperziae; taxonomy. †These authors contributed equally to this work. The DDBJ/EMBL/GenBank accession number for the 16S rRNA gene sequence of strain CPCC 204077T is KX527846. Two supplementary tables and one supplementary figure are available with the online Supplementary Material. 002284 ã 2017 IUMS
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Ai et al., Int J Syst Evol Microbiol 2017;67:4210–4215
0.25 g, K2HPO4 0.5 g, agar 15.0 g, tap water 1000 ml, pH 7.5) was employed as the isolation medium. Aztreonam (25 mg l 1), actidione (25 mg l 1) and potassium dichromate (30 mg l 1) were added to the medium to prevent the growth of Gram-stain-negative bacteria and fungi that might be present. After 3 weeks of incubation at 28 C, the visible colonies were picked and streaked onto PYG medium (l 1) (peptone 3.0 g, yeast extract 5.0 g, glycerol 10.0 ml, betaine hydrochloride 1.25 g, sodium pyruvate 1.25 g, agar 15 g) and incubating at 28 C to obtain the separated colonies. The purified culture was maintained on PYG agar at 4 C and as glycerol suspensions (20 %, v/v) at –80 C. Growth of the strain CPCC 204077T was tested at 28 C for 4–6 weeks on PYG medium, YS medium, Marine Agar (Difco), TSA medium, R2A Agar (Difco) and nutrient agar [11]. Growth conditions of strain CPCC 204077T were tested using PYG medium, incubating the cultures for up to 3 weeks. Different temperatures for growth (4, 10, 15, 20, 28, 30, 32, 35, 37, 40 and 45 C) were tested on PYG agar [pH 7.0, NaCl 0 % (w/v)]. NaCl tolerance was tested with different NaCl concentrations [0, 1, 2, 3, 5, 7 and 10 % (w/ v)] on PYG agar (28 C, pH 7.0). The pH range for growth (4.0–11.0, at intervals of 1.0 pH unit) was observed in PYG broth (28 C, 14–28 days) using the buffer system described by Xu et al. [12]. The oxidase activity was detected using API oxidase reagent (bioMerieux) according to the manufacturer’s instructions. Catalase activity was detected by observing the production of oxygen bubbles with addition of a drop of hydrogen peroxide solution (3 %, v/v). Metabolic characters were examined using Biolog GEN III (MicroPlate), API 50CH, API 20NE and API ZYM test kits (bioMerieux) according to the manufacturer’s instructions. Results were evaluated after incubation at 28 C for 48–96 h. Other physiological tests of the strain were examined according to previously described procedures [13]. The Gram reaction was tested by the standard Gram-staining method and observed using light microscopy (Axio Scope. A1 Vario; Zeiss). The morphology of cells was observed by light microscopy and confirmed using scanning electron microscopy (Quanta 200; FEI) with gold-coated dehydrated specimens of 7-day culture from PYG agar. Cells of strain CPCC 204077T were aerobic, Gram-stainpositive. Scanning electron microscopic observations revealed irregular coccoid to short rod-shaped cells that varied in size (0.4–0.61.1–1.2 µm) on PYG agar (Fig. 1). Strain CPCC 204077T grew well on PYG medium, while moderate growth occurred on YS medium, TSA medium, nutrient agar, and no growth was observed on Marine Agar (Difco) and R2A Agar (Difco). Colonies on PYG medium after a week at 28 C were circular, convex and smooth with ivory white to pale yellow colour and approximately 1.0 mm in diameter. Growth of strain CPCC 204077T was observed at 10–32 C, pH 5.0–8.0, in the presence of 0–1 % (w/v) NaCl. The optimum growth occurred at 28–30 C, pH 7.0, with the absence of NaCl. The strain hydrolysed gelatin and
Fig. 1. Scanning electron micrograph of strain CPCC 204077T grown on PYG agar for 7 days at 28 C. Bar, 4 µm.
urea but not CM-cellulose and starch. Peptonization of milk, nitrate reduction and H2S production tests were negative. Detailed physiological and biochemical characteristics of strain CPCC 204077T are given in the species description. Molecular systematic and chemotaxonomic studies of the strain CPCC 204077T were conducted with cells after cultivation in shake flasks on a rotary shaker (150 r.p.m.) using PYG broth at 28 C to its logarithmic phase, except that the mass used for cellular fatty acid analysis was harvested from TSB. The diagnostic sugars in the whole-cell hydrolysates of strain CPCC 204077T were examined using TLC as described by Lechevalier and Lechevalier [14, 15]. A peptidoglycan sample was prepared following the procedure described by Schleifer and Kandler [16]. Amino acids and peptides in cell-wall hydrolysates were analysed according to the method described by Schumann [17]. The polar lipids were examined by two-dimensional TLC and identified using previously described procedures [18]. Menaquinones were extracted according to Collins et al. [19], and analysed by HPLC [3]. Cellular fatty acids were extracted, methylated and analysed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions [20, 21]. MIDI Sherlock Version 6.0 and the ACTIN1 database were employed in this analysis. The peptidoglycan amino acid composition of strain CPCC 204077T was found to contain Lys (0.8), Ala (1.9), Ser (0.9), Glu (1.0), Asp (1.0) and Mur (1.3) (molar ratios in parentheses), and the enantiometric analysis revealed the ratio of amino acids as L-Lys (1.4), D-Ala (0.4), L-Ala (1.3), L-Ser (0.8), D-Asp (1.0) and D-Glu (1.0) (approximate molar ratios in parentheses). The peptides of mild hydrolysate detected were L-Ala-D-Glu, L-Lys-L-Ser, L-Lys-D-Ala and DAla-L-Lys-L-Ser. Based on these results, it is concluded that the peptidoglycan of strain CPCC 204077T was A4a (LLys-L-Ser-D-Asp). Galactose, glucose, rhamnose and ribose
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Ai et al., Int J Syst Evol Microbiol 2017;67:4210–4215
were detected in the whole-cell hydrolysates. The polar lipids consisted of a large amount of diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE), and a small amount of phosphatidylglycerol (PG), phosphatidylinositol (PI), one phospholipid (PL) and one glycolipid (GL) (Fig. S1, available in the online Supplementary Material). The only menaquinone was MK-8(H4). The predominant cellular fatty acid (>10 %) was iso-C16 : 0 (58.7 %) (Table S1). For analysis of the 16S rRNA gene sequence of strain CPCC 204077T, genomic DNA extraction, amplification and sequencing of purified products were carried out as described by Li et al. [22]. The 16S rRNA gene sequence of the isolate was compared with available 16S rRNA gene sequences from GenBank using the BLAST program and the EzTaxon-e server (http://www.ezbiocloud.net) [23] to determine an approximate phylogenetic affiliation. Multiple alignments with sequences of the most closely related taxa and calculations of levels of sequence similarity were carried out using MEGA version 5.0 [24]. Phylogenetic trees were inferred using the neighbour-joining method [25] with Knuc values [26, 27] and complete deletion gaps, and the maximum-parsimony [28] and maximum-likelihood [29] methods. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein [30] with 1000 replicates. The G+C content of the genomic DNA was determined using the thermal denaturation method [31]
using a UV-1700 spectrophotometer (SHIMADZU) equipped with a DCW-2008 thermo bath, with Calidifontibacter indicus JCM 16038T as a reference. The G+C content in the genomic DNA of strain CPCC 204077T was detected as 71.0 mol%. The almost full-length 16S rRNA gene sequence (1513 bp) of strain CPCC 204077T was obtained. BLAST search results showed that the novel isolate exhibited the highest similarities with members of the family Dermacoccaceae, such as Branchiibius hedensis Mer 29717T (95.0 %) and Calidifontibacter indicus PC IW02T (95.0 %), followed by Barrientosiimonas humi 39T (94.9 %) and Demetria terragena HKI 0089T (94.7 %). In the phylogenetic tree based on the 16S rRNA gene sequences of all genera within the family Dermacoccaceae, strain CPCC 204077T formed a distinct lineage alongside the genus Branchiibius (Fig. 2), standing for a genus position, which verified that strain CPCC 204077T should be assigned to a novel taxon in the family Dermacoccaceae. The analysis of 16S rRNA signature nucleotides demonstrated that strain CPCC 204077T shared the family-specific signature nucleotide pattern at positions 140–223 (C–G), 144–178 (C–G), 248–276 (C–G), 258–268 (G–C), 379–384 (C–G), 407–435 (A–U, G– C), 502–543 (A–U), 586–755 (C–G), 602–636 (C–G), 610 (R), 612–628 (Y–G), 615–625 (G–C), 616–624 (G–Y), 630 (Y), 668–738 (A–U), 839–847 (U–A), 863 (U) and 1133– 1141 (A–U), which was defined for the family
100 Barrientosiimonas humi DSM 24617T (JF346171) 100 Barrientosiimonas endolithica NBRC 110608T (LM994041)
0.01 99
Barrientosiimonas marina DSM 21415T (HE792770) Demetria terragena HKI 0089T (Y14152)
64
T 100 Branchiibius hedensis Mer 29717 (NR_112988) Branchiibius cervicis PAGU 1247T (AB594446)
92
Allobranchiibius huperziae CPCC 204077T (KX527846) Flexivirga alba ST13T (AB539735)
93
Rudaeicoccus suwonensis HOR6-4T (GQ241687) Luteipulveratus mongoliensis MN07-A0370T (AB468971) 89 100
Kytococcus aerolatus 02-St-019/1T (FM992368) Kytococcus schroeteri DSM 13884T (AJ297722) Kytococcus sedentarius DSM 20547T (CP001686)
Dermacoccus nishinomiyaensis DSM 20448T (X87757) 100 100 68 100
Dermacoccus abyssi MT1.1T (AY894323) Dermacoccus barathri MT2.1T (AY894328) Dermacoccus profundi MT2.2T (AY894329)
Calidifontibacter indicus PC IW02T (EF187228) Yimella lutea YIM 45900T (FJ528304) Streptomyces ambofaciens ATCC 23877T (AB184182)
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences showing the relationship of strain CPCC 204077T to representatives of the family Dermacoccaceae. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony methods. Bootstrap values (those above 50 %) are shown as percentages of 1000 replicates. Bar, 1 nt substitution per 100 nt.
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Gal, Glc, Rha, Rib MK-8(H4)
Whole-cell sugars
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DNA G+C content (mol %)
Polar lipids
Major fatty acids
Major menaquinone(s)
(1.4), (0.4), L-Ala (1.3), L-Ser (0.8), D-Glu (1.0), D-Asp (1.0)
L-Lys
DPG, PG, PE, PI, GL, PL 71.0
i-C16 : 0
D-Ala
ai-C17 : 0, ai-C17 : 1!9c, i-C16 : 0 DPG, PG, PGL, PI, GL, PL 68.4
MK-8(H4)
Gal, Glc
(1.6), (1.4), L-Ser (1.0), Gly (0.4), D-Glu (2.0), D-Ala (0.9) Gal, Man, Rha, Rib, Glc MK-8(H2), MK-8(H4) C17 : 1!8c, i-C16 : 0, C18 : 1 !9c DPG, PG, PI, PL 68
D-Ser
L-Lys
Ser–DAsp
L-Ser–
ND
ND
L-Lys–L-
L-Lys–
D-Asp
3
2
1
Amino acid composition (ratio) of peptidoglycan
Interpeptide bridge (A4a)
Characteristics
i-C16 : 0, i-C16 : 1 H, ai-C17 : 0 DPG, PG, PI, PIM, PS 77
MK-8(H4)
ND
ND
DPG, PG, PE, PI, PL 66
MK-8 (H4) C18 : 1, C18 : 0, C17 : 0
ND
Lys (0.8), Ala (1.8), Asp (1.2), Glu (1.0), Ser (0.8)
L-Lys–LSer–D-Asp
L-Lys–L-Gly–L-
Ser–D-Asp
5
4
MK-8 (H2) i-C15 : 0, i-C17 : 0, i-C17 : 1 DPG, PG, PI 66.8
Gal
L-Lys–LSer1–2–DGlu or L-Lys–LSer1–2–LAla–DGlu Ala (3.0), Ser (0.9), Glu (2.3), Lys (1.0)
6
67.4
PG
i-C16 : 0
DGlu (2.0), L-Ser (1.8), Gly (1.1), Ala (1.8), L-Lys (0.7) Gal, Glc, Rib MK-8(H4)
ND
7
66–69
DPG, PG, PI
MK-8, MK9, MK-10 i-C17 : 1, ai-C17 : 0
ND
Ala (1.6), Gly (0.1), Glu (2.5), Lys (1.0)
Glu2
L-Lys–
8
68.2
Gal, Glc, Man, Rha, Rib MK-8(H4), MK-8(H6) i-C16 : 0, ai-C17 : 0, i-C16 : 1 H, C17 : 1!9c, C17 : 0 10-Me DPG, PG, PI
L-Lys (0.8), Ala (2.7), Asp (0.6), Glu (1.0), Ser (0.9)
ND
9
MK-8(H4), MK-8(H6) ai-C17 : 0, 10-Me C18 : 0, 10-Me C17 : 0 DPG, PI, APL, PL, AL 65
Glc, Rib
L-Ala (1.7), Gly (1.1), Thr (0.9), D-Glu (2.2), L-Lys (1.0)
D-Glu
L-Thr–
L-Lys–
10
i-C15 : 0, ai-C15 : 0, ai-C17 : 0 DPG, PI, GlcN-PL, PL 65.8
MK-8(H4)
Gal, Fuc
Ala (1.6), Gly (1.0), Ser (0.7), Asp (0.8), Glu (1.0), Lys (1.2)
D-Asp
L-Ser–
L-Lys–
11
Genera: 1, Allobranchiibius gen. nov. CPCC 204077T (data from this study); 2, Barrientosiimonas [9]; 3, Branchiibius [6]; 4, Calidifontibacter [7]; 5, Demetnia [3]; 6, Dermacoccus [2]; 7, Flexivirga [8]; 8, Kytococcus [2]; 9, Luteipulveratus [4]; 10, Rudaeicoccus [10]; 11, Yimella [5]. ND, no data available; Gal, galactose; Glc, glucose; Man, mannose; Rha, rhamnose; Rib, ribose; Fuc, fucose; ai, ante-isomethyl branched; i, iso-methyl branched; Me, methyl branched; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PGL, phosphoglycolipid; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannoside; GL, glycolipid; GlcN-PL, glucosamine-containing phospholipid; PL, unknown phospholipid; PS, phosphatidylserine; AL, unknown aminolipid; APL, unknown aminophospholipid.
Table 1. Differentiating characteristics of strain CPCC 204077T and phylogenetically related genera of the family Dermacoccaceae
Ai et al., Int J Syst Evol Microbiol 2017;67:4210–4215
Ai et al., Int J Syst Evol Microbiol 2017;67:4210–4215
Dermacoccaceae [1]. Additionally, strain CPCC 204077T possessed its own signature nucleotide pattern distinguished from other recognized genera as summarized in Table S2. Chemotaxonomically, it is quite clear that strain CPCC 204077T differs from other members of the family Dermacoccaceae even though they all share a number of common characteristics. In the cellular fatty acid profile, strain CPCC 204077T shared the common major fatty acid of iso-C16 : 0 with its close phylogenetic neighbours. MK-8(H4) was detected in the menaquinone system, without MK-8(H2). One glycolipid was found in the polar lipid extracts, while mannose was not detected in its cellular sugar composition (Table 1). These data support the differentiation of strain CPCC 204077T from other genera in the family Dermacoccaceae. Therefore, on the basis of the phenotypic and phylogenetic distinctiveness presented above, strain CPCC 204077T is proposed to represent a novel species of a new genus in the family Dermacoccaceae, for which the name Allobranchiibius huperziae gen. nov., sp. nov., is proposed.
DESCRIPTION OF ALLOBRANCHIIBIUS GEN. NOV. Allobranchiibius (Al.lo.bran.chi.i¢bi.us. Gr. adj. allos another, the other, different; N.L. masc. n. Branchiibius a bacterial genus name; N.L. masc. n. Allobranchiibius the other Branchiibius, referring to the fact that it is phylogenetically related to Branchiibius). Gram-stain-positive, irregular coccoid to short rod-shaped cells that vary in size (0.4–0.61.1–1.2 µm), are non-sporeforming and non-motile. The peptidoglycan type is A4a with an L-Lys–L-Ser–D-Asp interpeptide bridge. Galactose, glucose, rhamnose and ribose are detected in whole-cell sugars. The polar lipids are composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, one unidentified phospholipid and one unidentified glycolipid. The menaquinone was MK-8 (H4). The major cellular fatty acid was iso-C16 : 0. The DNA G+C content of the type strain of the type species is 71.0 mol%. The signature nucleotide pattern is 127 : 234 (A– A), 185 : 192 (C–C), 187 (C), 199 : 218 (U–U), 200 : 217 (U– U), 203 : 214 (G–C), 1012 : 1017 (A–A), 1014 (C), 1015 (A), 1025 : 1036 (U–U), 1026 : 1035 (U–G), 1027 : 1034 (U–G), 1090 (A), 1245 : 1292 (A–U), 1267 (C) and 1450 (A). Phylogenetically, the genus is a member of the family Dermacoccaceae. The type species is Allobranchiibius huperziae.
NaCl concentration and temperature range for growth are pH 5.0–8.0, 0–1 % NaCl (w/v) and 10–32 C, with optimum growth at pH 7.0, 0 % NaCl (w/v) and 28–30 C, respectively. Colonies on PYG medium after a week at 28 C are circular, convex and smooth with ivory white to pale yellow colour. Positive for catalase and hydrolysis of gelatin, Tween 40 and urea, while negative for hydrolysis of starch and CM-cellulose, oxidase, peptonization of milk, H2S production and nitrate reduction. According to API ZYM enzyme assays, positive for acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase lipase (C8), esterase (C4), leucine arylamidase, lipase (C14), N-acetyl-b-glucosaminidase, naphthol-AS-B1-phosphohydrolase, trypsin, valine arylamidase, a-chymotrypsin, a-galactosidase, a-glucosidase, bglucuronidase, b-galactosidase, but not a-fucosidase and a-mannosidase. Produces acid from arbutin, D-arabitol, Dfructose, D-glucose, D-ribose, D-sucrose (sucrose), trehalose, D-xylose, erythritol, aesculin ferric citrate, glycerol, potassium 2-ketogluconate, potassium 5-ketogluconate, potassium gluconate and xylitol. Can utilize aztreonam, Dgalactose, glycerol and L-rhamnose as sole carbon sources. The G+C content of the type strain is 71.0 mol%. The type strain is CPCC 204077T (=NBRC 110719T=DSM 29531T), and was isolated from a medicinal plant Huperzia serrata (Thunb.) collected from Sichuan Province, China. Funding information This research was supported by the National Infrastructure of Microbial Resources (NIMR-2015-3), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-2-002) and the National Natural Science Foundation of China (NSFC 81173026; NSFC 31670010). Acknowledgements The authors thank the Institute of Microbiology, Chinese Academy of Sciences, for assistance in the cell morphology observations using scanning electron microscopy. Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical statement This research did not contain any studies with animals performed by any of the authors. References 1. Stackebrandt E, Schumann P. Description of Bogoriellaceae fam. nov., Dermacoccaceae fam. nov., Rarobacteraceae fam. nov. and Sanguibacteraceae fam. nov. and emendation of some families of the suborder Micrococcineae. Int J Syst Evol Microbiol 2000;50: 1279–1285.
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